Patent Grant
8268605
The present invention relates to compositions and methods utilizing DNA polymerase enzymes with reduced discrimination for non-conventional nucleotides. The enzymes of the invention are useful in many applications calling for the detectable labeling of nucleic acids and are particularly useful in DNA sequencing applications.
Detectable labeling of nucleic acids is required for many applications in molecular biology, including applications for research as well as clinical diagnostic techniques. A commonly used method of labeling nucleic acids uses one or more unconventional nucleotides and a polymerase enzyme that catalyzes the template-dependent incorporation of the unconventional nucleotide(s) into the newly synthesized complementary strand.
The ability of a DNA polymerase to incorporate the correct deoxynucleotide is the basis for high fidelity DNA replication in vivo. Amino acids within the active site of polymerases form a specific binding pocket that favors the placement of the correct complementary nucleotide opposite the template nucleotide. If a mismatched nucleotide, ribonucleotide, or nucleotide analog fills that position, the precise alignment of the amino acids contacting the incoming nucleotide may be distorted into a position unfavorable for DNA polymerization. Because of this, the unconventional nucleotides or nucleotide analogs used to label DNA tend to be incorporated into the elongated strand less efficiently than do the standard deoxynucleotide triphosphates (dNTPs; the so-called “standard” dNTPs include deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP)).
The reduced efficiency with which unconventional nucleotides are incorporated by the polymerase increases the amount of the unconventional nucleotide necessary for DNA labeling. The reduced efficiency of incorporation of a particular nucleotide can also adversely affect the performance of techniques or assays, such as DNA sequencing, that depend upon unbiased incorporation of unconventional nucleotides for homogeneous signal strength.
The identity and exact arrangement of the amino acids of a DNA polymerase that contact an incoming nucleotide triphosphate determine the nature of the nucleotides, both conventional and unconventional, that may be incorporated by that polymerase enzyme. Changes in the exact placement of the amino acids that contact the incoming nucleotide triphosphate at any stage of binding or chain elongation can dramatically alter the polymerase's capacity for utilization of unusual or unconventional nucleotides. Sometimes changes in distant amino acids can influence the incorporation of nucleotide analogs due to indirect global or structural effects. Polymerases with increased capacity to incorporate nucleotide analogs are useful for labeling DNA or RNA strands with nucleotides modified with signal moieties such as dyes, reactive groups or unstable isotopes.
In addition to labeled nucleotides, an extremely important class of modified nucleotides is the dideoxynucleotides. The so-called “Sanger” or “dideoxy” DNA sequencing method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463, which is incorporated herein by reference) relies upon the template-directed incorporation of nucleotides onto an annealed primer by a DNA polymerase from a mixture containing deoxy- and dideoxynucleotides. The incorporation of a dideoxynucleotide results in chain termination, the inability of the enzyme to catalyze further extension of that strand. Electrophoretic separation of reaction products results in a “ladder” of extension products wherein each extension product ends in a particular dideoxynucleotide complementary to the nucleotide opposite it in the template. The distance of the dideoxynucleotide analog from the primer is indicated by the length of the extension product. When four reactions, each containing one of the four dideoxynucleotide analogs ddA, ddC, ddG, or ddT (ddNTPs) are separated on the same gel, the sequence of the template may be read directly from the ladder patterns. Extension products may be detected in several ways, including for example, the inclusion of isotopically- or fluorescently-labeled primers, deoxynucleotide triphosphates or dideoxynucleotide triphosphates in the reaction.
Fluorescent labeling has the advantages of faster data collection, since detection may be performed while the gel is running, and longer reads of sequence data from a single reaction and gel. Further, fluorescent sequence detection has allowed sequencing to be performed in a single reaction tube containing four differentially-labeled fluorescent dye terminators (the so-called dye-terminator method, Lee et al., 1992, Nucleic Acids Res. 20: 2471, incorporated herein by reference).
A desirable quality of a polymerase useful for DNA sequencing is improved incorporation of dideoxynucleotides. Improved incorporation of dideoxynucleotides can make processes such as DNA sequencing more cost effective by reducing the requirement for expensive radioactive or fluorescent dye-labeled dideoxynucleotides. Moreover, unbiased dideoxynucleotide incorporation provides improved signal uniformity, leading to increased accuracy of base determination. The even signal output further allows subtle sequence differences caused by factors like allelic variation to be detected. Allelic variation, which produces two different half strength signals at the position of relevance, can easily be concealed by the varied signal strengths caused by polymerases with non-uniform ddNTP utilization.
Incorporation of ribonucleotides by the native form of DNA polymerase is a rare event. Mutants that incorporate higher levels of ribonucleotides can be used for applications such as sequencing by partial ribosubstitution. In this system, a mixture of ribonucleotides and deoxynucleotides corresponding to the same base are incorporated by the mutant polymerase (Barnes, 1978 J. Mol. Biol. 119:83-99). When the ribosequencing reactions are exposed to alkaline conditions and heat, fragmentation of the extended strand occurs. If the reactions for all four bases are separated on a denaturing acrylamide gel, they produce a sequencing ladder. there is a need in the art for polymerase mutants with higher utilization of ribonucleotides for this alternative method of sequencing.
Alternatively, the incorporation of ribonucleotides followed by alkaline hydrolysis could be utilized in a system that requires random cleavage of DNA molecules such as DNA shuffling ((Stemmer, 1994, Nature, 370: 389-391) which has also been called molecular breeding, sexual PCR and directed evolution).
Another desirable quality in a DNA labeling enzyme is thermal stability. DNA polymerases exhibiting thermal stability have revolutionized many aspects of molecular biology and clinical diagnostics since the development of the polymerase chain reaction (PCR), which uses cycles of thermal denaturation, primer annealing, and enzymatic primer extension to amplify DNA templates. The prototype thermostable DNA polymerase is Taq polymerase, originally isolated from the thermophilic eubacterium Thermus aquaticus. So-called “cycle sequencing” reactions using thermostable DNA polymerases have the advantage of requiring smaller amounts of starting template relative to conventional (i.e., non-cycle) sequencing reactions.
There are three major families of DNA polymerases, termed families A, B and C. The classification of a polymerase into one of these three families is based on structural similarity of a given polymerase to E. coli DNA polymerase I (Family A), II (Family B) or III (family C). As examples, Family A DNA polymerases include, but are not limited to Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase) and bacteriophage T7 DNA polymerase; Family B DNA polymerases, formerly known as α-family polymerases (Braithwaite and Ito, 1991, Nuc. Acids Res. 19:4045), include, but are not limited to human α, δ and ε DNA polymerases, T4, RB69 and φ29 bacteriophage DNA polymerases, and Pyrococcus furiosus DNA polymerase (Pfu polymerase); and family C DNA polymerases include, but are not limited to Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase III α and ε subunits (listed as products of the dnaE and dnaQ genes, respectively, by Brathwaite and Ito, 1993, Nucleic Acids Res. 21: 787). An alignment of DNA polymerase protein sequences of each family across a broad spectrum of archaeal, bacterial, viral and eukaryotic organisms is presented in Braithwaite and Ito (1993, supra), which is incorporated herein by reference.
The term used to describe the tendency of DNA polymerases to not to carry the incorporation of unnatural nucleotides into the nascent DNA polymer is “discrimination”. In Family A DNA polymerases, the effective discrimination against incorporation of dideoxynucleotide analogs is largely associated with a single amino acid residue. The majority of enzymes from the Family A DNA polymerases have a phenylalanine (phe or F) residue at the position equivalent to F762 in E. coli Klenow fragment of DNA polymerase and demonstrate a strong discrimination against dideoxynucleotides. A few polymerases (e.g. T7 DNA polymerase) have a tyrosine (tyr or Y) residue at the corresponding position and exhibit relatively weak discrimination against dideoxynucleotides. Family A polymerases with tyrosine at this position readily incorporate dideoxynucleotides at levels equal to or only slightly different from the levels at which they incorporate deoxynucleotides. Conversion of the tyrosine or phenylalanine residues in the site responsible for discrimination reverses the dideoxynucleotide discrimination profile of the Family A enzymes (Tabor and Richardson, 1995, Proc. Natl. Acad. Sci. USA 92:6449).
Among the thermostable DNA polymerases, a mutant form of the Family A DNA polymerase from Thermus aqaticus, known as AmpliTaq FS® (Perkin Elmer), contains a F667Y mutation at the position equivalent to F762 of Klenow DNA polymerase and exhibits increased dideoxynucleotide uptake (i.e., reduced discrimination against ddNTPs) relative to the wild-type enzyme. The reduced discrimination for dideoxynucleotide uptake makes it more useful for fluorescent and labeled dideoxynucleotide sequencing than the wild-type enzyme.
The F667Y mutant of Taq DNA polymerase is not suited, however, for use with fluorescein-labeled dideoxynucleotides, necessitating the use of rhodamine dye terminators. Rhodamine dye terminators that are currently utilized with Taq sequencing reactions, however, stabilize DNA secondary structure, causing compression of signal. Efforts to eliminate compression problems have resulted in systems that use high amounts of the nucleotide analog deoxyinosine triphosphate (dITP) in place of deoxyguanosine triphosphate. While incorporation of (dITP) reduces the compression of the signal, the presence of dITP in the reaction produces additional complications including lowered reaction temperatures and increased reaction times. Additionally, the use of rhodamine dyes in sequencing requires undesirable post-reaction purification (Brandis, 1999 Nuc. Acid Res. 27:1912).
Family B DNA polymerases exhibit substantially different structure compared to Family A DNA polymerases, with the exception of the position of acidic residues involved in catalysis in the so-called palm domain (Wang et al., 1997, Cell 89:1087; Hopfner et al., 1999, Proc. Natl. Acad. Sci. USA 96:3600). The unique structure of Family B DNA polymerases may permit a completely different spectrum of interactions with nucleotide analogs, perhaps allowing utilization of analogs which are unsuitable for use with Family A DNA polymerases due to structural constraints. Thermostable Family B DNA polymerases have been identified in hyperthermophilic archaea. These organisms grow at temperatures higher than 901 C. and their enzymes demonstrate greater themostability (Mathur et al., 1992, Stratagies 5:11) than the thermophilic eubacterial Family A DNA polymerases. Family B polymerases from hyperthermophilic archaea may be well suited starting substrates for modification(s) to reduce discrimination against non-conventional nucleotides.
Although the crystal structures of three Family B DNA polymerases have been solved (Wang et al., 1997, supra; Hopfner, K.-P. et al., 1999, Proc. Natl. Acad. Sci. 96: 3600; Zhao, 1999, Structure Fold Des., 7:1189), the structures of DNA-polymerase or dNTP-polymerase co-complexes have not yet been reported. At present, identification of amino acid residues contributing to nucleotide analog discrimination can only be inferred from extrapolation to Family A-dNTP structures or from mutagenesis studies carried out with related Family B DNA polymerases (e.g., human polα, phage T4, phage φ29, T. litoralis DNA polymerase).
Sequence comparison of the Family B DNA polymerases indicate six conserved regions numbered I-VI (Braithwaite and Ito, 1993, supra). The crystal structure of bacteriophage RB69 DNA polymerase (Family B) proposed by Wang et al. (Wang et al., 1997, supra) shows that Y416 in region II (which corresponds to Y409 in the Family B DNA polymerase of Thermococcus species JDF-3) has the same position as Y115 in HIV reverse transcriptase (RT) and E710 in the Klenow fragment (Family A polymerases). Modeling of the dNTP and primer template complex in RB69 was carried out using the atomic coordinates of the reverse transcriptase-DNA cocrystal. This model predicts the RB69 Y416 packs under the deoxyribose portion of the dNTP. Tyrosine at this position has been implicated in ribose selectivity, contributing to polymerase discrimination between ribonucleotides and deoxribonucleotides in mammalian reverse transcriptases (Y115) (Gao et al., 1997, Proc. Natl. Acad. Sci. USA 94:407; Joyce, 1994, Proc. Natl. Acad. Sci. USA 94:1619) and in Family A DNA polymerases where modification of the corresponding invariable glutamate residue (E710) reduces discrimination against ribonucleotides (Gelfand et al., 1998, Pat. No. EPO823479; Astatke et al, 1998, Proc. Natl. Acad. Sci. USA 96:3402).
Mutagenesis studies done in Family B DNA polymerases also implicate the region containing the analogous Y in region II in dNTP incorporation and ribose selectivity. Mutations at the corresponding Y865 in human DNA polymerase α affect polymerase fidelity and sensitivity to dNTP nucleotide inhibitors such as AZT-TP, which has a bulky 3′-azido group in place of the 3′-OH group, BuPdGTP, which contains a butylphenyl group attached to the amino group at the C-2 position in the guanine base of dGTP (resulting in a bulkier and more hydrophobic purine base nucleotide) and aphidicolin, a competitive inhibitor of pyrimidine deoxynucleotide triphosphate. Interestingly, the mutants showed no difference in their uptake of ddCTP (Dong et al., 1993, J. Biol. Chem. 268: 26143). Additionally, mutants of bacteriophage T4 DNA polymerase, which have converted L412 to methionine (M) or isoleucine (I) just one amino acid before the analogous Y (Y411), show extreme and mild sensitivity, respectively, to the inorganic pyrophosphate analog phosphonoacetic acid (PAA). Alterations in PAA sensitivity have been shown to predict polymerase interactions with nucleotide analogs. L412 in T4 DNA polymerase corresponds to L410 in Thermococcus species JDF-3 DNA polymerase. The L412M T4 DNA polymerase mutant was inhibited with 50-fold less ddGTP than wild-type polymerase while the Kms for dGTP was similar. As stated by the authors in that study, “[d]espite the sensitivity of the L412M DNA polymerase to ddGTP, there was no difference found in the incorporation of ddNTPs by wild-type and L412M DNA polymerase.” (Reha-Krantz et al., 1993, J. Virol. 67:60). In bacteriophage φ29, mutations in region II (LYP where Y is analogous to Thermococcus species JDF3 DNA polymerase Y409) produce mixed results when challenged with PAA; P255S was hypersensitive to PAA while L253V was shown to be less sensitive than the wild-type enzyme (Blasco et al., 1993, J. Biol. Chem. 268: 24106). These data support the role of the LYP region (region II) in polymerase-nucleotide interactions, but improved incorporation of ddNTPs was not achieved in these references.
In another study, extensive mutation of region II in the archaeal Family B DNA polymerase from Thermococcus litoralis DNA polymerase (VENT™ polymerase, New England Biolabs) was performed. In that study, 26 different site-directed mutants were made for the sole intent of examining nucleotide analog discrimination (Gardner and Jack, 1999, Nucleic Acids Res. 27: 2545). Site-directed mutagenesis of VENT™ DNA polymerase demonstrated that three mutations at Y412 (which corresponds to JDF-3 DNA polymerase Y409) could alter nucleotide binding (Gardner and Jack, 1999, supra). Y412V was most significant with a 2 fold increase in dideoxynucleotide incorporation and a 200 fold increase in the incorporation of ribonucleotide ATP. The mutation Y412F showed no change in analog incorporation.
Region III of the Family B polymerases (also referred to as motif B) has also been demonstrated to play a role in nucleotide recognition. This region, which corresponds to AA 487 to 495 of JDF-3 Family B DNA polymerase, has a consensus sequence KX3NSXYG (Jung et al., 1990, supra; Blasco et al., 1992, supra; Dong et al., 1993, J. Biol. Chem. 268:21163; Zhu et al., 1994, Biochem. Biophys. Acta 1219:260; Dong and Wang, 1995, J. Biol. Chem. 270:21563), and is functionally, but not structurally (Wang et al., 1997, supra), analogous to KX3(F/Y)GX2YG in helix O of the Family A DNA polymerases. In Family A DNA polymerases, such as the Klenow fragment and Taq DNA polymerases, the O helix contains amino acids that play a major role in dNTP binding (Astatke et al., 1998, J. Mol. Biol. 278:147; Astatke et al., 1995, J. Biol. Chem. 270:1945; Polesky et al., 1992, J. Biol. Chem 267:8417; Polesky et al., 1990, J. Biol. Chem. 265:14579; Pandey et al., 1994, J. Biol. Chem. 269:13259; Kaushik et al., 1996, Biochem. 35:7256). Specifically, helix O contains the F (F763 in the Klenow fragment; F667 in Taq) which confers ddNTP discrimination in Family A DNA polymerases (KX3(F/Y)GX2YG) (Tabor and Richardson, 1995, supra).
Directed mutagenesis studies in region III of VENT™ DNA polymerase also targeted an alanine analogous to A485 of the Thermococcus species JDF-3 DNA polymerase. These mutants (A→C, A→S, A→L, A→I, A→F and A→V) exhibited a range of specific activities from 0.12 to 1.2 times the polymerase activity of the progenitor enzyme (Gardner and Jack, 1999, Nucl. Acids Res. 27:2545). The dideoxynucleotide incorporation ranged from 4 to 15 times the unmutated enzyme. Interestingly, the mutant with the highest dideoxynucleotide incorporation (15×) had a specific activity of only 0.12× of the original enzyme.
Site-directed mutagenesis studies on the Family B DNA polymerase from Thermococcus barossii modified each residue independently in the sequence ILANSF (SEQ ID NO:49), which corresponds to AA residues 488-493 of the JDF-3 DNA polymerase, to tyrosine (Reidl et al., U.S. Pat. No. 5,882,904). That study indicated that an L489Y mutant exhibits approximately 3 times greater incorporation of dideoxynucleotides relative to an enzyme bearing the wild-type leucine residue at this site.
One area of active research involves the use of nucleic acid arrays, often referred to as nucleic acid or DNA “chips”, in the simultaneous analyses of multiple different nucleic acid sequences. Many of these applications, such as those described in U.S. Pat. No. 5,882,904 (Reidl et al., issued Mar. 16, 1999) will benefit from DNA polymerases exhibiting reduced discrimination against non-conventional nucleotides, particularly fluorescently-labeled non-conventional nucleotides. Applications being addressed in the chip format include DNA sequencing and mutation detection, among others. For example, the “mini-sequencing” methods (e.g., Pastinen et al., 1997, Genome Res. 7: 606; Syvanen, 1999, Human Mutation 13: 1-10) and the arrayed primer extension (APEX) mutation detection method (Shumaker et al., 1996, Hum. Mutat. 7: 346) and methods like them can benefit from DNA polymerases with reduced discrimination against fluorescently-labeled or other non-conventional nucleotides. There is a need in the art for a non-discriminating DNA polymerase for use in chip or gel based mini-sequencing systems. Such a system would advantageously permit detection of multiplexed single nucleotide polymorphisms (SNPs) and allow for quantitative genotyping. Identification of sequence variation permits the diagnosis and treatment of genetic disorders, predisposition to multifactorial diseases, and sensitivity to new or existing pharmaceutical products.
With the completion of the human genome project, considerable attention is now focused on analyzing genetic variations between individuals, and specifically, single nucleotide polymorphisms (SNPs) which have been estimated to occur one in every 1000 bp (Halushka et al., 1999). The importance of SNPs is that they serve as genetic markers that enable identification of disease related loci (Lai et al., 1998). They can also be used to investigate the underlying cause of genetic diseases and could eventually help pave the way to personalized medicine.
Current assays used in SNP detection include hybridization to allele-specific oligonucleotide (ASO) probes (Saiki et al., 1989), oligonucleotide ligation assay (OLA) (Landegren et al., 1988), restriction fragment length polymorphism (RFLP) (Shi et al., 2001), TaqMan assay (Livak et al., 1995), molecular beacon assay (Tyagi et al., 1998), and primer extension assay (Tyagi et al., 1998; Gilles et al., 1999; Fu et al., 1998) on a variety of platforms including gel electrophoresis (Chen et al., 1997), MALDI-TOF mass spectrometry (Fu et al., 1998), solid phase minisequencing (Syvanen et al., 1990), semiconductor microchips (Gilles et al., 1999), and flow cytometric analysis (Taylor et al., 2001).
The principle of minisequencing is to anneal primers immediately adjacent to the SNP positions to be analyzed and to extend these primers with ddNTPs complementary to the SNP (Syvanen et al., 1990, hereby incorporated as reference) using a DNA polymerase that readily incorporates ddNTPs. Minisequencing is unique since it is based on the high accuracy (high specificity) of polymerase mediated nucleotide incorporation reactions rather than the thermostability of matched and mismatched species which affects most other SNP detection methods. Thus, compared to hybridization-based methods, minisequencing is insensitive to small variations in reaction conditions, temperature, and to flanking DNA sequence. Moreover, minisequencing allows discrimination between homozygous and heterozygous genotypes (Chen et al., 1997). These characteristics are important in multiplexing and/or high throughput SNP detection. With the completion of the genome project and considerable interest in high throughput SNP detection, a significant market exists for enzymes that efficiently incorporate ddNTPs and dye labeled-ddNTPs in single base extension assays (minisequencing).
DNA polymerases constitute a core component of minisequencing protocols. Efficient ddNTP and dye-ddNTP incorporation and high fidelity are essential characteristics of minisequencing enzymes. Commercially available DNA polymerases that are suitable for sequencing and minisequencing have been derived from either Taq (Taq F667Y mutants such as ThermoSequenase and AmpliTaqFS) or bacteriophage T7 DNA polymerase (Sequenase), which are both family A DNA polymerases. A tyrosine (Y) residue in the nucleotide binding pocket of T7 (native) or Taq (engineered F667Y mutant) DNA polymerase confers efficient ddNTP incorporation (Tabor et al., 1995). In two recent mutagenesis studies employing archaeal (family B) DNA polymerases, mutations were identified that reduced ddNTP discrimination; however, the archaeal DNA polymerase mutants incorporated ddNTPs less efficiently than the Taq F667Y mutant(Gardner et al., 1999; Evans et al., 2000).
There is a need in the art for DNA polymerases with reduced discrimination against unconventional nucleotides. There is particularly a need in the art for thermostable DNA polymerases exhibiting reduced discrimination against dideoxynucleotides, and further, for DNA polymerases exhibiting reduced discrimination against fluorescently labeled dideoxynucleotides.
The present invention relates to compositions and methods utilizing DNA polymerase enzymes exhibiting reduced discrimination against non-conventional nucleotides. Enzymes with this quality are useful in many applications calling for the detectable labeling of nucleic acids and are particularly useful in DNA sequencing applications.
The invention further relates to a Family B DNA polymerase having one or more mutations at a site or sites corresponding to L408, P410, S345, and/or A485 of SEQ ID NO: 2, or a fragment thereof which retains the ability to direct the template-dependent polymerization of nucleic acid. The invention also encompasses mutants and modified versions (e.g., reversibly inactivated versions of a Family B polymerase prepared, for example, by chemical modification or antibody complexing) of a Family B polymerase mutated at sites corresponding to L408, P410 and or A485 of SEQ ID NO: 2.
In one embodiment, the DNA polymerase has a dual mutation comprising comprising a serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2; and a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
The invention encompasses purified thermostable DNA polymerase having an amino acid sequence presented in SEQ ID NO: 2 from residue 1 to 776.
In one embodiment, the thermostable DNA polymerase is isolated from Thermococcus species JDF-3.
In another embodiment, the thermostable polymerase is isolated from a recombinant organism transformed with a vector that codes for the expression of Thermococcus species JDF-3 DNA polymerase.
The invention further encompasses a recombinant vector comprising the nucleotide sequence presented in SEQ ID NO: 1.
The invention further encompasses an isolated recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof.
The invention further encompasses an isolated recombinant DNA polymerase from Thermococcus species JDF-3 that is 3′ to 5′ exonuclease deficient.
In one embodiment, the isolated recombinant DNA polymerase of has an aspartic acid to threonine or alanine mutation at the amino acid corresponding to D141 of SEQ ID NO: 2 or a glutamic acid to alanine mutation at the amino acid corresponding to E143 of SEQ ID NO: 2.
In another embodiment, the isolated recombinant DNA polymerase has an aspartic acid to threonine or alanine mutation at the amino acid corresponding to D141 of SEQ ID NO: 2 and a glutamic acid to alanine mutation at the amino acid corresponding to E143 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having reduced discrimination against non-conventional nucleotides.
In one embodiment, the DNA polymerase is a Family B DNA polymerase.
In another embodiment, the DNA polymerase further comprises a mutation selected from the group consisting of: a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2; a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2; a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2; and an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having the alanine to threonine mutation at the site corresponding to A485 of SEQ ID NO:2 further comprising a mutation selected from the group consisting of: a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2; a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2; and a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having the a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2, further comprising of serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2
In another embodiment, the DNA polymerase has reduced discrimination against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides, ribonucleotides and conjugated nucleotides.
In another embodiment, conjugated nucleotide is selected from the group consisting of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.
The invention further encompasses an isolated recombinant Family B DNA polymerase comprising an alanine to threonine mutation at the site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides relative to the wild-type form of that polymerase.
In one embodiment, the Family B DNA polymerase is 3′ to 5′ exonuclease deficient.
In another embodiment, the Family B DNA polymerase has a mutation at an amino acid corresponding to D141 or E143 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has an aspartic acid to threonine or alanine mutation at a site corresponding to D141 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has a glutamic acid to alanine mutation at a site corresponding to E143 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has a glutamic acid to alanine mutation at a site corresponding to E143 of SEQ ID NO: 2 and has an aspartic acid to threonine or alanine mutation at the amino acid corresponding to D141 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase is thermostable.
In another embodiment, the Family B DNA polymerase is archaeal.
In another embodiment, the Family B DNA polymerase comprises a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2, further having a serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2, and may further comprise a mutation at a site corresponding to T604 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a tyrosine to cysteine mutation at a site corresponding to Y497 of SEQ ID NO: 2, and may further comprise an isoleucine to valine mutation at a site corresponding to I630 of SEQ ID NO:2.
In another embodiment, the Family B DNA polymerase comprises a glutamic acid to lysine mutation at a site corresponding to E645 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a glutamic acid to lysine mutation at a site corresponding to E578 of SEQ ID NO: 2, and may further comprise an arginine to methionine mutation at a site corresponding to R465 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to glutamine mutation at a site corresponding to L396 of SEQ ID NO: 2, and may further comprise a mutation at a site corresponding to V401, N424, P569, E617, or V640 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a serine to asparagene mutation at a site corresponding to S651 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to proline mutation at a site corresponding to L396 of SEQ ID NO: 2, and may further comprise a mutation at a site corresponding to E459 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to proline mutation at a site corresponding to L456 of SEQ ID NO: 2, and may further comprise a mutation at a site corresponding to E658 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2, and may further comprise a mutation at a site corresponding to V437, or L478 of SEQ ID NO: 2. The L408H mutation was isolated both in the dideoxynucleotide and the dye-dideoxynucleotide screens described herein.
In another embodiment, the Family B DNA polymerase comprises an tyrosine to asparagine mutation at a site corresponding to Y496 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has reduced discrimination against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides, ribonucleotides and conjugated nucleotides.
In another embodiment, the conjugated nucleotide is selected from the group consisting of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.
In another embodiment, an isolated recombinant DNA polymerase having reduced discrimination against non-conventional nucleotides or an isolated recombinant Family B DNA polymerase comprising an alanine to threonine mutation at the site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides relative to the wild-type form of that polymerase further comprises a mutation at an amino acid residue in the polymerase that corresponds to a mutation selected from the group consisting of: a Y to V mutation at amino acid 409 of SEQ ID NO:2; an A to C, S, L, I, F, or V mutation at amino acid 485 of SEQ ID NO: 2; a Y to S mutation at amino acid 494 of SEQ ID NO: 2; a Y to L mutation at amino acid 496 of SEQ ID NO: 2; and an A to Y mutation at amino acid 490 of SEQ ID NO: 2.
In another embodiment, an isolated recombinant DNA polymerase having reduced discrimination against non-conventional nucleotides or an isolated recombinant Family B DNA polymerase comprising an alanine to threonine mutation at the site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides relative to the wild-type form of that polymerase further comprises a mutation at an amino acid of the polymerase corresponding to one of amino acids 483 to 496, inclusive, of SEQ ID NO: 2.
In one embodiment, the mutation is at an amino acid of the polymerase corresponding to one of amino acids 485, 490, 494, or 496 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant Family B DNA polymerase comprising an alanine to threonine mutation at an amino acid corresponding to A485T of SEQ ID NO: 2 and at least one substitution in the polymerase of an amino acid corresponding to L408, Y409, or P410, respectively, of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant Family B DNA polymerase comprising an amino acid other than A at an amino acid of the polymerase corresponding to A485 of SEQ ID NO: 2, and at least one substitution in the polymerase of an amino acid corresponding to L408, Y409, or P410, respectively, of SEQ ID NO: 2.
The invention further encompasses a recombinant vector comprising a nucleic acid sequence encoding the Family B DNA polymerase.
The invention further encompasses a method of labeling a complementary strand of DNA, the method comprising the step of contacting a template DNA molecule with a recombinant Family B DNA polymerase from Thermococcus species JDF-3, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides, and a non-conventional nucleotide, under conditions and for a time sufficient to permit the DNA polymerase to synthesize a complementary DNA strand and to incorporate the non-conventional nucleotide into the synthesized complementary DNA strand.
The invention further encompasses a method of labeling a complementary strand of DNA, the method comprising the step of contacting a template DNA molecule with a recombinant Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides, and a non-conventional nucleotide, under conditions and for a time sufficient to permit the DNA polymerase to synthesize a complementary DNA strand and to incorporate the non-conventional nucleotide into the synthesized complementary DNA strand.
In one embodiment, the recombinant Family B DNA polymerase is 3′ to 5′ exonuclease deficient.
In another embodiment, the recombinant Family B polymerase comprises a leucine to histidine mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprises a leucine to phenylalanine mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprises a proline to leucine mutation at a site corresponding to amino acid P410 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprises an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 comprises a leucine to histidine mutation at an amino acid corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 comprises a leucine to phenylalanine mutation at an amino acid corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 comprises a proline to leucine mutation at an amino acid corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has reduced discrimination against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides, ribonucleotides, and conjugated nucleotides.
The invention further encompasses a method of sequencing DNA comprising the steps of contacting a DNA strand to be sequenced with a sequencing primer, a recombinant Family B DNA polymerase from Thermococcus species JDF-3, wherein the DNA polymerase has reduced discrimination against non-conventional nucleotides, and a chain-terminating nucleotide analog, under conditions that permit the DNA polymerase to synthesize a complementary DNA strand, and to incorporate nucleotides into the synthesized complementary DNA strand, wherein incorporation of a chain-terminating nucleotide analog results in the termination of chain elongation, such that the nucleotide sequence of the template DNA strand is determined.
The invention further encompasses a method of sequencing DNA comprising the steps of contacting a DNA strand to be sequenced with a sequencing primer, a recombinant Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408, S345 or P410 of SEQ ID NO: 2, where the DNA polymerase has reduced discrimination against non-conventional nucleotides, and a chain-terminating nucleotide analog, under conditions that permit the DNA polymerase to synthesize a complementary DNA strand, and to incorporate nucleotides into the synthesized complementary DNA strand, wherein incorporation of a chain-terminating nucleotide analog results in the termination of chain elongation, such that the nucleotide sequence of the template DNA strand is determined.
In one embodiment, the recombinant DNA polymerase is deficient in 3′ to 5′ exonuclease activity.
In another embodiment, the recombinant Family B polymerase has a leucine to histidine mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has a leucine to phenylalanine mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has a proline to leucine mutation at a site corresponding to amino acid P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2, further having a serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the chain-terminating nucleotide analog is a dideoxynucleotide.
In another embodiment, the dideoxynucleotide is detectably labeled.
In another embodiment, the dideoxynucleotide is fluorescently labeled.
In another embodiment, the dideoxynucleotide is labeled with a moiety selected from the group consisting of fluorescein and rhodamine.
The invention also encompasses a kit for performing the methods disclosed herein.
The invention also encompasses methods of making a recombinant DNA polymerase as disclosed here, comprising culturing a host cell containing a nucleic acid sequence encoding said polymerase under conditions which permit production of said DNA polymerase.
The invention encompasses a mixture of a mutant DNA polymerase described herein and another DNA polymerase such as Taq DNA polymerase (preferably the mutant form, F667Y). Such a mixture is useful in that it may increase signal uniformity generated from polymerization of a labeled nucleotide into a synthetic nucleotide.
The invention provides a composition for identifying a nucleotide at a given position of a template DNA molecule, the composition comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein the first primer anneals to the immediate 3′ of the nucleotide at the given position of the template DNA molecule.
In one embodiment, the Family B DNA polymerase is a JDF-3 DNA polymerase.
In a preferred embodiment, the JDF-3 DNA polymerase has a sequence of SEQ ID NO: 2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.
In a preferred embodiment, the JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of: D141A or D141T, E143A, L408H or L408F, A485T, and P410L.
In a preferred embodiment, the JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.
Preferably, the Family B DNA polymerase in the above embodiments is deficient in 3′ to 5′ exonuclease activity.
The above embodiments of the present invention may further comprise at least one chain-terminating nucleotide analog, wherein the chain-terminating nucleotide analog is incorporated into the first primer by the Family B DNA polymerase in a template-dependent manner.
In a preferred embodiment, at least one chain-terminating nucleotide analog is labeled with a first detectable label.
In another preferred embodiment, more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.
Preferably, the chain-terminating nucleotide analog in the above embodiments is a dideoxynucleotide.
More preferably, the dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.
In a preferred embodiment, the first primer is labeled with a second detectable label.
In another preferred embodiment, the first and second detectable labels generate a signal for identifying the nucleotide at the given position of the template DNA molecule.
The above compositions for identifying a nucleotide may further comprise a second primer.
In one embodiment, the first primer is labeled with a second detectable label and the second primer is labeled with a third detectable label, the second and third detectable labels generate a signal for identifying the nucleotide at the given position of the template DNA molecule.
In a preferred embodiment, the second primer anneals to the immediate 5′ of the nucleotide at the given position of the template DNA molecule.
In one embodiment, the above composition further comprises a DNA ligase.
In a preferred embodiment, each above composition further comprises a reaction buffer for the Family B DNA polymerase.
In a preferred embodiment, the template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.
In another preferred embodiment, the first or second or third detectable label is one selected from the group consisting of: a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.
In another preferred embodiment, the first detectable label is a rhodamine label or a cyanine label.
The invention further provides each of the above compositions in kit format.
In a preferred embodiment, the kit further comprises a control template and/or at least one control primer.
In a more preferred embodiment, the kit further comprises a control template and four control primers.
The invention provides a method of identifying a nucleotide at a given position of a template DNA molecule in a sample, the method comprising:
In a preferred embodiment, the Family B DNA polymerase is a JDF-3 DNA polymerase.
In another embodiment, the JDF-3 DNA polymerase has a sequence of SEQ ID NO: 2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.
In another embodiment, the JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of: D141A or D141T, E143A, L408H or L408F, A485T, and P410L.
In yet another embodiment, the JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.
Preferably, the Family B DNA polymerase is deficient in 3′ to 5′ exonuclcase activity.
In one embodiment, at least one chain-terminating nucleotide analog is labeled with a first detectable label.
In another embodiment, more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.
Preferably, the chain-terminating nucleotide analog is a dideoxynucleotide.
More preferably, the dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.
In one embodiment, the first primer is labeled with a second detectable label.
In another embodiment, the first and second detectable labels generate a signal for identifying the nucleotide at the given position of the template DNA molecule.
In a preferred embodiment, the template DNA molecule is the product of a polymerase chain reaction or a plasmid.
In a preferred embodiment, the method of the present invention further comprises removing PCR primers and dNTPs from the PCR product before step (a).
The invention provides a method of identifying a nucleotide at a given position of a template DNA molecule in a sample, the method comprising:
In a preferred embodiment, the first or second or third detectable label is one selected from the group consisting of: a radiolabel, a fluorescent label, a chemiluminescent label, a calorimetric label and an enzymatic label.
In another embodiment, the first detectable label is a rhodamine label or a cyanine label.
As used herein, “discrimination” refers to the tendency of DNA polymerase to not incorporate non-conventional nucleotides into a nascent DNA polymer. DNA polymerase has the ability to sense nucleotide structure, including but not limited to nucleotide base complementarity, and structural features of the sugar and heterocyclic base, thereby allowing DNA polymerase to preferentially utilize conventional deoxynucleotides rather than non-conventional nucleotides for incorporation into a nascent polymer. DNA polymerase strongly prefers to incorporate the conventional deoxynucleotides dATP, dCTP, dGTP and dTTP into DNA polymers; the polymerase is unlikely to progress with an unconventional nucleotide in its binding pocket.
As used herein, “reduced discrimination” refers to a reduction of at least 50% in the tendency of a DNA polymerase to exclude a non-conventional nucleotide from (that is, to not incorporate non-conventional nucleotides into) a nascent DNA polymer, relative to a parental or wild type DNA polymerase which does not exhibit reduced discrimination. The preference of DNA polymerase to incorporate the conventional deoxynucleotides dATP, dCTP, dGTP and TTP rather than non-conventional nucleotides into DNA polymers is thereby reduced compared to the natural level of preference, such that non-conventional nucleotides are more readily incorporated into DNA polymers by DNA polymerase. According to the invention, a polymerase exhibiting reduced discrimination will exhibit reduced discrimination against at least one non-conventional nucleotides, but may not exhibit reduced discrimination against all non-conventional nucleotides.
According to the invention, discrimination is quantitated by measuring the concentration of a non-conventional nucleotide required to inhibit the incorporation of the corresponding conventional nucleotide by 50%. This concentration is referred to herein as the “I50%” for a non-conventional nucleotide. Discrimination against a given non-conventional nucleotide is “reduced” if the I50% for that non-conventional nucleotide is reduced by at least two fold (50%) relative to an identical assay containing, in place of the mutant DNA polymerase, a parental DNA polymerase.
Alternatively, reduced discrimination may be quantitated by determining the amount of a non-conventional nucleotide (for example, a dideoxynucleotide, ribonucleotide, or cordycepin) required in a reaction with a mutant polymerase having reduced discrimination to generate a sequencing ladder identical to a sequencing ladder produced using the wild-type or parental enzyme. The sequencing ladder can be examined, for example, in the range of 1 to 400 bases from the primer terminus, and the ladders will be identical in the number of extension products generated as well as the lengths of extension products generated in the sequencing reaction. For this type of assay, a constant amount of dNTPs and varying amounts of non-conventional nucleotides are used to generate a sequencing ladder with both the wild-type (or parental) enzyme and the mutant polymerase (for ribonucleotides, a sequencing ladder is generated by alkali cleavage of the polymerization products). See Gardner & Jack, 1999, supra. A mutant exhibits reduced discrimination if it requires at least two-fold (50%) less, five-fold (80%) less, ten-fold (100%) less, etc. of the amount of the non-conventional nucleotide used by the wild-type or parental polymerase to produce a sequencing ladder identical (with respect to the number and length of extension products generated) to that generated by the wild-type or parental enzyme.
As used herein, the term “parental” or “progenitor” refers to a polymerase used as the starting material in generating a mutant polymerase having reduced discrimination. The term “parental” is meant to encompass not only a so-called “wild-type” enzyme as it occurs in nature, but also intermediate forms, for example, an exonuclease deficient enzyme that is used as the starting material for generating an enzyme with reduced discrimination against non-conventional nucleotides.
As used herein, “non-conventional nucleotide” refers to a) a nucleotide structure that is not one of the four conventional deoxynucleotides dATP, dCTP, dGTP, and dTTP recognized by and incorporated by a DNA polymerase, b) a synthetic nucleotide that is not one of the four conventional deoxynucleotides in (a), c) a modified conventional nucleotide, or d) a ribonucleotide (since they are not normally recognized or incorporated by DNA polymerases) and modified forms of a ribonucleotide. Non-conventional nucleotides include but are not limited to those listed in Table III, which are commercially available, for example, from New England Nuclear. Any one of the above non-conventional nucleotides may be a “conjugated nucleotide”, which as used herein refers to nucleotides bearing a detectable label, including but not limited to a fluorescent label, isotope, chemiluminescent label, quantum dot label, antigen, or affinity moiety.
As used herein, the term “cell”, “cell line” and “cell culture” can be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.
As used herein, the term “organism transformed with a vector” refers to an organism carrying a recombinant gene construct.
As used herein, “thermostable” refers to a property of a DNA polymerase, such that the enzyme active at elevated temperatures and is resistant to DNA duplex-denaturing temperatures in the range of about 93° C. to about 97° C. “Active” means the enzyme retains the ability to effect primer extension reactions when subjected to elevated or denaturing temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Elevated temperatures as used herein refer to the range of about 70° C. to about 75° C., whereas non-elevated temperatures as used herein refer to the range of about 35° C. to about 50° C.
As used herein, “archaeal” refers to an organism or to a DNA polymerase from an organism of the kingdom Archaea.
As used herein, “primer” refers to an oligonucleotide, whether natural or synthetic, which is substantially complementary (i.e., at least 7 out of 10, preferably 9 out of 10, more preferably 9 out of 10 bases are fully complementary) and can anneal to a complementary template DNA to form a duplex between the primer and the template DNA. A primer may serve as a point of initiation of nucleic acid synthesis by a polymerase following annealing to a DNA strand to be sequenced. A primer is typically a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer, but for DNA sequencing applications typically ranges from about 15 to about 40 nucleotides in length.
As used herein, “Family B DNA polymerase” refers to any DNA polymerase that is classified as a member of the Family B DNA polymerases, where the Family B classification is based on structural similarity to E. coli DNA polymerase II. The Family B DNA polymerases, formerly known as α-family polymerases, include, but are not limited to those listed as such in Table I.
As used herein, “Family A DNA polymerase” refers to any DNA polymerase that is classified as a member of the Family A DNA polymerases, where the Family A classification is based on structural similarity to E. coli DNA polymerase I. Family A DNA polymerases include, but are not limited to those listed as such in Table I.
As used herein, “3′ to 5′ exonuclease deficient” or “3′ to 5′ exo−” refers to an enzyme that substantially lacks the ability to remove incorporated nucleotides from the 3′ end of a DNA polymer. DNA polymerase exonuclease activities, such as the 3′ to 5′ exonuclease activity exemplified by members of the Family B polymerases, can be lost through mutation, yielding an exonuclease-deficient polymerase. As used herein, a DNA polymerase that is deficient in 3′ to 5′ exonuclease activity substantially lacks 3′ to 5′ exonuclease activity. “Substantially lacks” encompasses a complete lack of activity, or a “substantial” lack of activity. “Substantial” lack of activity means that the 3′ exonuclease activity of the mutant polymerase relative to the parental polymerase is 0.03%, and also may be 0.05%, 0.1%, 1%, 5%, 10%, or 20%, but is not higher than 50% of the 3′ exonuclease activity of the parental or wild type polymerase.
As used herein, “mutation” refers to a change introduced into a starting parental DNA sequence that changes the amino acid sequence encoded by the DNA. The consequences of a mutation include but are not limited to the creation of a new character, property, function, or trait not found in the protein encoded by the parental DNA.
As used herein, “wild-type” refers to the typical state of an organism, strain, gene, protein or characteristic as it occurs in nature. The wild-type is therefore the natural state that is distinguished from a mutant, which was derived from the wild type by introduction of change(s) to the wild-type.
As used herein, “corresponding” refers to sequence similarity in a comparison of two or more nucleic acids or polypeptides, where functionally equivalent domains or sub-sequences are identified; such functionally equivalent domains or sub-sequences or amino acids within such a domain or sub-sequence are said to “correspond”. That is, two or more sequences are compared through a comparative alignment analysis in which an entire sequence is examined for regions of sequence that are similar or identical, and thus regions likely to be functionally equivalent to regions from the other sequence(s) are identified.
As used herein in reference to comparisons of an amino acid, amino acid sequence, or protein domain, the term “similar” refers to amino acids or domains that although not identical, represent “conservative” differences. By “conservative” is meant that the differing amino acid has like characteristics with the amino acid in the corresponding or reference sequence. Typical conservative substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. In calculating the degree (most often as a percentage) of similarity between two polypeptide sequences, one considers the number of positions at which identity or similarity is observed between corresponding amino acid residues in the two polypeptide sequences in relation to the entire lengths of the two molecules being compared.
As used herein, the term “functionally equivalent” means that a given motif, region, or amino acid within a motif or region performs the same function with regard to the overall function of the enzyme as a motif, region or amino acid within a motif or region performs in another enzyme.
As used herein, “chain terminating nucleotide analog” refers to a nucleotide analog that once incorporated cannot serve as a substrate for subsequent extension by a DNA polymerase, thereby terminating the elongation of a DNA polymer by a DNA polymerase. Such a nucleotide analog typically lacks a hydroxyl group on its sugar moiety to which DNA polymerase can synthesize a phosphodiester bond with an incoming nucleotide. Chain terminating nucleotide analogs are a subset of non-conventional nucleotides, and include but are not limited to dideoxynucleotides.
As used herein, “detectably labeled” refers to a structural modification that incorporates a functional group (label) that can be readily detected by various means. Compounds that can be detectably labeled include but are not limited to nucleotide analogs. Detectable nucleotide analog labels include but are not limited to fluorescent compounds, isotopic compounds, chemiluminescent compound, quantum dot labels, biotin, enzymes, electron-dense reagents, and haptens or proteins for which antisera or monoclonal antibodies are available. The various means of detection include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
As used herein in reference to a polynucleotide or polypeptide, the term “isolated” means that a naturally occurring sequence has been removed from its normal cellular environment or is synthesized in a non-natural environment (e.g., artificially synthesized). Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide or polypeptide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide or non-polypeptide material, respectively, naturally associated with it.
As used herein, the term “recombinant” refers to a polynucleotide or polypeptide that is altered by genetic engineering (i.e., by modification or manipulation of the genetic material encoding that polynucleotide or polypeptide).
The invention encompasses full length mutant DNA polymerases, as described herein, as well as a functional fragment of a mutant polymerase, that is, a fragment of a DNA polymerase that is less than the entire amino acid sequence of the mutant polymerase and retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein.
As used herein, the term “complementary DNA strand” refers to that DNA molecule synthesized from a template DNA molecule by a DNA polymerase in a primer extension reaction.
As used herein, the term “template DNA molecule” refers to that strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction. A “template DNA molecule”, also refers to a template DNA strand whose sequence needs to be identified. The sequence may need to be identified for a single nucleotide at a given position of the template DNA molecule (i.e., by mini-sequencing) or for a fragment of or the whole DNA molecule (i.e., by sequencing). The term “sequence”, according to the present invention, refers to the identification of a nucleotide at a given position or at more than one position of a template DNA molecule.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.
The invention is based on the discovery of Family B DNA polymerases that bear one or more genetic alterations resulting in reduced discrimination against non-conventional nucleotides relative to their unmodified wild-type forms. All references described herein are incorporated by reference herein in their entirety.
Family B DNA Polymerase Exhibiting Reduced Discrimination Against Non-Conventional Nucleotides:
A. DNA Polymerases Useful According to the Invention
According to the invention, DNA polymerases of Family B may be mutated to generate enzymes exhibiting reduced discrimination against non-conventional nucleotides. Table I includes a non-limiting list of known DNA polymerases categorized by family.
E. coli DNA polymerase 1
Streptococcus pneumoniae DNA polymerase I
Thermus aquaticus DNA polymerase I
Thermus flavus DNA polymerase I
Thermotoga maritima DNA polymerase I
E. coli DNA polymerase II
Thermococcus litoralis DNA polymerase (Vent)
Pyrococcus furiosus DNA polymerase
Sulfolobus solfataricus DNA polymerase
Thermococcus gorgonarius DNA polymerase
Sulfolobus acidocaldarius
Pyrodictium occultum
Methanococcus voltae
S. cerevisiae DNA polymerase (alpha)
S. pombe DNA polymerase I (alpha)
Drosophila melanogaster DNA polymerase (alpha)
Trypanosoma brucei DNA polymerase (alpha)
S. cerevisiae DNA polymerase III (delta)
S. pombe DNA polymerase III (delta)
Plasmodiun falciparum DNA polymerase (delta)
S. cerevisiae DNA polymerase II (epsilon)
S. cerevisiae DNA polymerase Rev3
Choristoneura biennis DNA polymerase
Autographa california nuclear polymerase virus
Lymantria dispar nuclear polyhedrosis virus DNA
B. Plasmids
The starting sequences for the generation of Family B DNA polymerases according to the invention may be contained in a plasmid vector. A non-limiting list of cloned Family B DNA polymerases and their GenBank Accession numbers are listed in Table II.
Desulfurococcus sp. Tok. (SEQ ID NO: 78)
Plasmids acceptable for the expression of modified forms of Family B DNA polymerases may be selected from a large number known in the art by one of skill in the art. A plasmid vector for expression of a modified DNA polymerase according to the invention will preferably comprise sequences directing high level expression of a DNA polymerase, and will more preferably comprise sequences directing inducible, high level expression of a DNA polymerase As one example of an inducible high level expression system, plasmids placing a modified DNA polymerase coding sequence according to the invention under the control of a bacteriophage T7 promoter may be introduced to bacteria containing an inducible T7 RNA polymerase gene within their chromosome. Induction of the T7 RNA polymerase gene subsequently induces high level expression of the T7 promoter-driven modified DNA polymerase gene (see for example, Gardner & Jack, Nucleic Acids Res. 27: 2545).
C. Mutagenesis
The cloned wild-type form of a Family B DNA polymerase may be mutated to generate forms exhibiting reduced discrimination against non-conventional nucleotides by a number of methods.
First, methods of random mutagenesis which will result in a panel of mutants bearing one or more randomly-situated mutations exist in the art. Such a panel of mutants may then be screened for those exhibiting reduced discrimination relative to the wild-type polymerase (see “Methods of Evaluating Mutants for Reduced Discrimination”, below). An example of a method for random mutagenesis is the so-called “error-prone PCR method”. As the name implies, the method amplifies a given sequence under conditions in which the DNA polymerase does not support high fidelity incorporation. The conditions encouraging error-prone incorporation for different DNA polymerases vary, however one skilled in the art may determine such conditions for a given enzyme. A key variable for many DNA polymerases in the fidelity of amplification is, for example, the type and concentration of divalent metal ion in the buffer. The use of manganese ion and/or variation of the magnesium or manganese ion concentration may therefore be applied to influence the error rate of the polymerase.
Second, there are a number of site-directed mutagenesis methods known in the art which allow one to mutate a particular site or region in a straightforward manner. There are a number of kits available commercially for the performance of site-directed mutagenesis, including both conventional and PCR-based methods. Examples include the EXSITE™ PCR-Based Site-directed Mutagenesis Kit available from Stratagene (Catalog No. 200502; PCR based) and the QUIKCHANGE™ Site-directed mutagenesis Kit from Stratagene (Catalog No. 200518; non-PCR-based), and the CHAMELEON® double-stranded Site-directed mutagenesis kit, also from Stratagene (Catalog No. 200509).
Older methods of site-directed mutagenesis known in the art relied upon sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods one annealed a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerized the complement of the template starting from the 3′ end of the mutagenic primer. The resulting duplexes were then transformed into host bacteria and plaques were screened for the desired mutation.
More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.
The protocol described below accomodates these considerations through the following steps. First, the template concentration used is approximately 1000-fold higher than that used in conventional PCR reactions, allowing a reduction in the number of cycles from 25-30 down to 5-10 without dramatically reducing product yield. Second, the restriction endonuclease DpnI (recognition target sequence: 5-Gm6ATC-3, where the A residue is methylated) is used to select against parental DNA, since most common strains of E. coli Dam methylate their DNA at the sequence 5-GATC-3. Third, Taq Extender is used in the PCR mix in order to increase the proportion of long (i.e., full plasmid length) PCR products. Finally, Pfu DNA polymerase is used to polish the ends of the PCR product prior to intramolecular ligation using T4 DNA ligase.
The method is described in detail as follows:
PCR-based Site Directed Mutagenesis of the 3′-5′ Exonuclease domain
Plasmid template DNA (approximately 0.5 pmole) is added to a PCR cocktail containing: 1× mutagenesis buffer (20 mM Tris HCl, pH 7.5; 8 mM MgCl2; 40 ug/ml BSA); 12-20 pmole of each primer (one of skill in the art may design a mutagenic primer as necessary, giving consideration to those factors such as base composition, primer length and intended buffer salt concentrations that affect the annealing characteristics of oligonucleotide primers; one primer must contain the desired mutation, and one (the same or the other) must contain a 5′ phosphate to facilitate later ligation), 250 uM each dNTP, 2.5 U Taq DNA polymerase, and 2.5 U of Taq Extender (Available from Stratagene; See Nielson et al. (1994) Strategies 7: 27, and U.S. Pat. No. 5,556,772). The PCR cycling is performed as follows: 1 cycle of 4 min at 94° C., 2 min at 50° C. and 2 min at 72° C.; followed by 5-10 cycles of 1 min at 94° C., 2 min at 54° C. and 1 min at 72° C. The parental template DNA and the linear, PCR-generated DNA incorporating the mutagenic primer are treated with DpnI (10 U) and Pfu DNA polymerase (2.5U). This results in the DpnI digestion of the in vivo methylated parental template and hybrid DNA and the removal, by Pfu DNA polymerase, of the non-template-directed Taq DNA polymerase-extended base(s) on the linear PCR product. The reaction is incubated at 37° C. for 30 min and then transferred to 72° C. for an additional 30 min. Mutagenesis buffer (115 ul of 1×) containing 0.5 mM ATP is added to the DpnI-digested, Pfu DNA polymerase-polished PCR products. The solution is mixed and 10 ul are removed to a new microfuge tube and T4 DNA ligase (2-4 U) is added. The ligation is incubated for greater than 60 min at 37° C. Finally, the treated solution is transformed into competent E. coli according to standard methods.
D. Non-Conventional Nucleotides Useful According to the Invention
There is a wide variety of non-conventional nucleotides available in the art. Any or all of them are contemplated for use with a DNA polymerase of the invention. A non-limiting list of such non-conventional nucleotides is presented in Table III.
Additional non-conventional nucleotides useful according to the invention include, but are not limited to 7-deaza-dATP, 7-deaza-dGTP, 5′-methyl-2′-deoxycytidine-5′-triphosphate. Further non-conventional nucleotides or variations on those listed above are discussed by Wright & Brown, 1990, Pharmacol. Ther. 47: 447. It is specifically noted that ribonucleotides qualify as non-conventional nucleotides, since ribonucleotides are not generally incorporated by DNA polymerases. Modifications of Family B DNA polymerases that result in the ability, or enhanced ability, of the polymerase to incorporate labeled or unlabeled ribonucleotides are specifically contemplated herein.
E. Methods of Evaluating Mutants for Reduced Discrimination
Random or site-directed mutants generated as known in the art or as described herein and expressed in bacteria may be screened for reduced discrimination against non-conventional nucleotides by several different assays. In one method, Family B DNA polymerase proteins expressed in lytic lambda phage plaques generated by infection of host bacteria with expression vectors based on, for example, Lambda ZapII®, are transferred to a membrane support. The immobilized proteins are then assayed for polymerase activity on the membrane by immersing the membranes in a buffer containing a DNA template and the unconventional nucleotides to be monitored for incorporation.
Mutant polymerase libraries may be screened using a variation of the technique used by Sagner et al (Sagner, G., Ruger, R., and Kessler, C. (1991) Gene 97:119-123). For this approach, lambda phage clones are plated at a density of 10-20 plaques per square centimeter. Proteins present in the plaques are transferred to filters and moistened with polymerase screening buffer (50 mM Tris (pH 8.0), 7 mM MgCl2, 3 mM β-ME). The filters are kept between layers of plastic wrap and glass while the host cell proteins are heat-inactivated by incubation at 65° C. for 30 minutes. The heat-treated filters are then transferred to fresh plastic wrap and approximately 35 μl of polymerase assay cocktail are added for every square centimeter of filter. The assay cocktail consists of 1× cloned Pfu (cPfu) magnesium free buffer (1× buffer is 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 100 ug/ml bovine serum albumin (BSA), and 0.1% Triton X-100; Pfii Magnesium-free buffer may be obtained from Stratagene (Catalog No. 200534)), 125 ng/ml activated calf thymus or salmon sperm DNA, 1.29 μCi/ml α-33P ddNTP or dideoxynucleotides (at a dNTP:dye-ddNTP ratio of 1:15). Initial screening was done in the presence of MnCl2, but the preferred method was to screen in 1×Taq Polymerase buffer (1.5 mM MgCl2) The filters are placed between plastic wrap and a glass plate and then incubated at 65° C. for one hour, and then at 70° C. for one hour and fifteen minutes. Filters are then washed three times in 2×SSC for five minutes per wash before rinsing twice in 100% ethanol and vacuum drying. Filters are then exposed to X-ray film (approximately 16 hours), and plaques that incorporate label are identified by aligning the filters with the original plate bearing the phage clones. Plaques identified in this way are re-plated at more dilute concentrations and assayed under similar conditions to allow the isolation of purified plaques.
In assays such as the one described above, the signal generated by the label is a direct measure of the activity of the polymerase with regard to that particular unconventional nucleotide or combination of unconventional nucleotides used in the assay. Unconventional nucleotides corresponding to all four conventional nucleotides may be included in the reactions, or, alternatively, only one unconventional nucleotide may be included to assess the effect of the mutation(s) on utilization of a given unconventional nucleotide. One approach is to use unconventional nucleotides corresponding to all four nucleotides in a first screen to identify clones that incorporate more than a reference wild-type clone, and then to monitor the incorporation of individual unconventional nucleotides in a subsequent screen. In the preferred screening mode, only the dideoxynucleotides and dideoxynucleotide analogs of ddATP, ddCTP, and ddTTP would be used since ddGTP is not discriminated against by some DNA polymerases and increases the background signal of any screen
In order to screen for clones with enhanced ability to incorporate dideoxynucleotides, clones identified in first screens utilizing only dideoxynucleotides may then be characterized by their sensitivity to low levels of each of the four dideoxynucleotides in a DNA polymerase nucleotide incorporation assay employing all four dNTPs, a; H-TTP tracer, and a low level of each ddNTP. Since incorporation of dideoxynucleotides stops DNA chain elongation, superior ability to incorporate dideoxynucleotides diminishes the incorporation of tritium labeled deoxynucleotides relative to wild-type DNA polymerase. Comparisons of ddNTP concentrations that bring about 50% inhibition of nucleotide incorporation (I50%) can be used to compare ddNTP incorporation efficiency of different polymerases or polymerase mutants. Comparisons of I50% values for ddATP, ddCTP, ddGTP, and ddTTP can be used to identify mutants with reduced selectivity for particular bases. Such mutants would be expected to produce more uniform DNA sequencing ladders.
In order to measure incorporation of individual ddNTPs, cocktails are prepared which consist of varying concentrations of the ddNTP of interest, and a total of 200 μM of each nucleotide triphosphate. For example, the incorporation of ddATP by wild type JDF-3 polymerase may be measured at 0, 40, 80, 120 and 160 μM ddATP. In these reactions, dATP concentrations would be adjusted to 200, 160, 120, 80, and 40 μM, respectively, so that the total amount of adenine nucleotide triphosphate is 200 μM. In comparison, mutants may be assayed using ddATP concentrations of 0, 5, 10, and 20 μM ddATP, and adjusted dATP concentrations of 200, 195, 190, and 180 μM, respectively (dATP+ddATP=200 μM). Additional cocktails are prepared to similarly measure ddCTP, ddGTP, and ddTTP incorporation.
Incorporation of nucleotides under the concentration parameters described above may be measured in extension reactions by adding, for example, 1 μl of appropriately diluted bacterial extract (i.e., heat-treated and clarified extract of bacterial cells (see Example 1, part M) expressing a cloned polymerase or mutated cloned polymerase) to 10 ul of each nucleotide cocktail, followed by incubation at 72° C. for 30 minutes. Extension reactions are quenched on ice, and then 5 μl aliquots are spotted immediately onto DE81 ion-exchange filters (2.3 cm; Whatman #3658323). Unincorporated label is removed by 6 washes with 2×SCC (0.3M NaCl, 30 mM sodium citrate, pH 7.0), followed by a brief wash with 100% ethanol. Incorporated radioactivity is then measured by scintillation counting. Reactions that lack enzyme are also set up along with sample incubations to determine “total cpms” (omit filter wash steps) and “minimum cpms” (wash filters as above).
Cpms bound is proportional to the amount of polymerase activity present per volume of bacterial extract. The volume of bacterial extract (generally about 0.25-1 μl) which brings about incorporation of approximately 10,000 cpms is determined for use in subsequent nucleotide analog incorporation testing.
Genes for mutant DNA polymerases generated by random mutagenesis may be sequenced to identify the sites and number of mutations. For those mutants comprising more than one mutation, the effect of a given mutation may be evaluated by introduction of the identified mutation to the exo− progenitor gene by site-directed mutagenesis in isolation from the other mutations borne by the particular mutant. Screening assays of the single mutant thus produced will then allow the determination of the effect of that mutation alone.
F. Expression of Mutated Family B DNA Polymerase According to the Invention
Methods known in the art may be applied to express and isolate the mutated forms of Family B DNA polymerase according to the invention. Many bacterial expression vectors contain sequence elements or combinations of sequence elements allowing high level inducible expression of the protein encoded by a foreign sequence. For example, as mentioned above, bacteria expressing an integrated inducible form of the T7 RNA polymerase gene may be transformed with an expression vector bearing a mutated DNA polymerase gene linked to the T7 promoter. Induction of the T7 RNA polymerase by addition of an appropriate inducer, for example, isopropyl-β-D-thiogalactopyranoside (IPTG) for a lac-inducible promoter, induces the high level expression of the mutated gene from the T7 promoter (see Gardner & Jack, 1999, supra).
Appropriate host strains of bacteria may be selected from those available in the art by one of skill in the art. As a non-limiting example, E. coli strain BL-21 is commonly used for expression of exogenous proteins since it is protease deficient relative to other strains of E. coli. BL-21 strains bearing an inducible T7 RNA polymerase gene include WJ56 and ER2566 (Gardner & Jack, 1999, supra). For situations in which codon usage for the particular polymerase gene differs from that normally seen in E. coli genes, there are strains of BL-21 that are modified to carry tRNA genes encoding tRNAs with rarer anticodons (for example, argU, ileY, leuW, and proL tRNA genes), allowing high efficiency expression of cloned protein genes, for example, cloned archaeal enzyme genes (several BL21-CODON PLUS™ cell strains carrying rare-codon tRNAs are available from Stratagene, for example).
There are many methods known to those of skill in the art that are suitable for the purification of a modified DNA polymerase of the invention. For example, the method of Lawyer et al. (1993, PCR Meth. & App. 2: 275) is well suited for the isolation of thermostable DNA polymerases expressed in E. coli, as it was designed originally for the isolation of Taq polymerase. Alternatively, the method of Kong et al. (1993, J. Biol. Chem. 268: 1965, incorporated herein by reference) may be used, which employs a heat denaturation step to destroy host proteins, and two column purification steps (over DEAE-Sepharose and heparin-Sepharose columns) to isolate highly active and approximately 80% pure thermostable DNA polymerase. Further, as detailed in Example 1, part N, below, DNA polymerase mutants may be isolated by an ammonium sulfate fractionation, followed by Q Sepharose and DNA cellulose columns, or by adsorption of contaminants on a HiTrap Q column, followed by gradient elution from a HiTrap heparin column.
G. Preparation of Thermococcus species JDF-3 Thermostable DNA Polymerase With Reduced Discrimination
To prepare thermostable Family B polymerases which exhibit reduced discrimination for dideoxynucleotide triphosphates (ddNTPs), the DNA sequence encoding a 3′ to 5′ exonuclease-deficient (D141A) Family B polymerase from the hyperthermophilic archaeon Thermococcus species JDF-3 was subjected to random mutagenesis using “error-prone PCR” as described herein, and cloned into the bacteriophage lambda Zap®II. The polymerase from JDF-3 was chosen due to superior processivity, polymerization rate and ddNTP incorporation relative to the Family B DNA polymerase from Pyrococcus furiosus (Pfu) (see Table IV, below). The library of mutants was plated on E. coli hosts and the proteins present in the lytic plaques were transferred to a solid support that was then immersed in a buffer containing DNA template and all four α-33p labeled dideoxynucleotides. Mutants that incorporated the labeled dideoxynucleotide produced signals that corresponded to their ability to incorporate the α-33P ddNTPs. Isolated clones were then characterized by their sensitivity to low levels of each of the four dideoxynucleotides in a DNA polymerase nucleotide incorporation assay employing all four dNTPs and a; H-TTP tracer. Since incorporation of dideoxynucleotides stops DNA chain elongation, superior ability to incorporate dideoxynucleotides diminishes the incorporation of tritium labeled deoxynucleotides. The unmutated progenitor DNA polymerase rarely incorporates dideoxynucleotides and is only 50% inhibited at high ddNTP levels (100-160 micromolar each ddNTP). The mutant enzymes show 50% inhibition at 5 to 40 micromolar concentrations of ddNTP and improved incorporation was observed for all four ddNTPs (ddATP, ddCTP, ddTTP and ddGTP; see Tables V and VI in Example 1, below).
The incorporation of non-conventional nucleotides was also evaluated through use of purified mutant polymerases in cycle sequencing, with α-33p labeled ddNTPs present at 0.021 μM and dNTPs present at 2.1 μM each. The mutants readily utilized all four dideoxynucleotides and produced sequencing ladders that compared favorably to Thermo Sequenase®, which uses an F667Y Taq DNA polymerase mutant (VanderHom et al., 1997, BioTechniques 22: 758).
The domains of relevance in 17 of the 40 purified mutants were sequenced. Most randomly mutated clones contained more than one mutation in the regions sequenced but all mutants contained mutations at one of three sites. Mutations predicted to confer an enhanced ddNTP uptake phenotype were introduced into the progenitor exonuclease deficient DNA polymerase sequence by site-directed mutagenesis to eliminate ancillary mutations which were not expected to contribute to the improved dideoxynucleotide uptake phenotype.
Sixteen of the seventeen JDF-3 DNA polymerase mutations were found in region II (motif A) on either side of the tyrosine in the consensus sequence 404 DxxSLYPSII 413. These mutations consisted of DFRSLYLSII (P410 L), DFRSHYPSII (L408H) and DFRSFYPSII (L408F). Therefore, the LYP motif of region II appears to be important in ddNTP discrimination in the JDF-3 Family B polymerase.
The prior art modification of the tyrosine corresponding to Y409 in JDF3 Family B DNA polymerase is recognized for its positioning in the nucleotide binding pocket. As shown herein, however, modification of the residues neighboring Y409 (L408H or L408F or P410L) had the unexpected effect of profoundly altering nucleotide binding, particularly with respect to ddNTP incorporation.
The only JDF-3 DNA polymerase mutation leading to enhanced incorporation of non-conventional nucleotides occurring outside of region II is an alanine (ala or A) to threonine (thr or T) conversion at position 485 in region III (A485T). This site is two residues upstream of KX3NSXYG (Jung et al., 1990, supra; Blasco et al., 1992, supra; Dong et al., 1993, J. Biol. Chem. 268:21163; Zhu et al., 1994, Biochem. Biophys. Acta 1219:260; Dong and Wang, 1995, J. Biol. Chem. 270:21563) (referred to as region III or motif B) which is functionally, but not structurally (Wang et al., 1997, supra), analogous to KX3(F/Y)GX2YG in helix O of the Family A DNA polymerases. In Family A DNA polymerases, such as the Klenow fragment and Taq DNA polymerases, the O helix contains amino acids that play a major role in dNTP binding (Astatke et al., 1998, J. Mol. Biol. 278:147; Astatke et al., 1995, J. Biol. Chem. 270:1945; Polesky et al., 1992, J. Biol. Chem 267:8417; Polesky et al., 1990, J. Biol. Chem. 265:14579; Pandey et al., 1994, J. Biol. Chem. 269:13259; Kaushik et al., 1996, Biochem. 35:7256). Specifically, helix O contains the F (F762 in the Klenow fragment; F667 in Taq) which confers ddNTP discrimination in Family A DNA polymerases (KX3(F/MY)GX2YG) (Tabor and Richardson, 1995, supra).
The effect of the A485T mutation on ddNTP incorporation in the JDF-3 DNA polymerase is surprising since the RB69 and Thermococcus gorgonarius crystal structures (Hopfner et al., 1999, supra) show it facing away from the proposed active site of the nucleotide binding surface. Moreover, the type of side chain conferring ribose selectivity in archaeal Family B DNA polymerases (A: small, non-polar) is different from that of the bulky, aromatic Y and F residues that dictate ddNTP discrimination in Family A DNA polymerases (Tabor and Richardson, 1995, supra). Additionally, this position (A485) is not well conserved among either DNA polymerase family and is not included in the consensus sequence for this domain (Braithwaite and Ito, 1993, supra), implying a lack of critical importance in dNTP recognition.
A JDF-3 double mutant was constructed that contains mutations P410L and A485T. In dideoxynucleotide cycle sequencing, the banding pattern intensity demonstrated by the double mutant was extremely uniform, suggesting little if any preference for any dNTP over its corresponding ddNTP (See
The following examples are offered by way of illustration only and are by no means intended to limit the scope of the claimed invention.
A. Cloning a DNA Polymerase Gene from Thermococcus Species JDF-3 DNA Polymerase
A Thermococcus species was cultured from submarine samples taken from the Juan de Fuca ridge. Genomic DNA was isolated and used to prepare a genomic DNA library in ZAP II (Stratagene) using standard procedures. The lambda library was plated on XL1-Blue MRF′ E. coli and screened for clones with DNA polymerase activity using a variation of the method described by Sagner et al. (Sagner, G., Ruger, R., and Kessler, C. (1991) Gene 97:119-123). Plaques containing active polymerase were cored and stored in SM buffer. Positive primary plaques were re-plated and re-assayed to allow purification of isolated clones. Secondary clones were excised according to the instructions provided with the ZAP II system (Stratagene), and the DNA sequence of the insert determined (
The translated amino acid sequence of the JDF-3 DNA polymerase is shown in
Recombinant JDF-3 DNA polymerase was purified as described below (see “Purification of JDF-3” (method 1)). The biochemical properties of JDF-3 DNA polymerase have been compared to those of other commercially available archaeal DNA polymerases. The results shown in Table IV and V indicated that, compared to other enzymes, JDF-3 exhibits higher processivity, a faster polymerization rate (Kcat), and a greater tendency to utilize ddNTPs. JDF-3 DNA polymerase was therefore chosen for development of a DNA sequencing enzyme.
aH. Kong, R. B. Kucera, and W. E Jack, J. Biol. Chem. 268, 1965 (1993).
B. Intein Removal from the Gene Encoding JDF-3 DNA Polymerase
By alignment to Family B DNA polymerase sequences, the JDF-3 DNA polymerase clone was found to contain an intein sequence (
C. Construction of a JDF-3 DNA Polymerase Mutant with Diminished 3′-5′ Exonuclease Activity
DNA polymerases lacking 3′-5′ exonuclease (proofreading) activity are preferred for applications requiring nucleotide analog incorporation (e.g., DNA sequencing) to prevent removal of nucleotide analogs after incorporation. The 3′-5′ exonuclease activity associated with proofreading DNA polymerases can be reduced or abolished by mutagenesis. Sequence comparisons have identified three conserved motifs (exo I, II, III) in the 3′-5′ exonuclease domain of DNA polymerases (reviewed V. Derbyshire, J. K. Pinsonneault, and C. M. Joyce, Methods Enzymol. 262, 363 (1995)). Replacement of any of the conserved aspartic or glutamic acid residues with alanine has been shown to abolish the exonuclease activity of numerous DNA polymerases, including archaeal DNA polymerases such as Vent (H. Kong, R. B. Kucera, and W. E. Jack, J. Biol. Chem. 268, 1965 (1993)) and Pfu (Stratagene, unpublished). Conservative substitutions lead to reduced exonuclease activity, as shown for mutants of the archaeal 9° N-7 DNA polymerase (M. W. Southworth, H. Kong, R. B. Kucera, J. Ware, H. Jannasch, and F. B. Perler, Proc. Natl. Acad. Sci. 93, 5281 (1996)).
JDF-3 DNA polymerase mutants exhibiting substantially reduced 3′-5′ exonuclease activity were prepared by introducing amino acid substitutions at the conserved 141D or 143E residues in the exo I domain. Using the CHAMELEON® Double-Stranded, Site-Directed Mutagenesis Kit (Stratagene), the following JDF-3 mutants were constructed: D141A, D141N, D141S, D141T, D141E and E143A.
To analyze JDF-3 mutant proteins, the DNA sequence encoding JDF-3 DNA polymerase was PCR amplified using primers GGG AAA CAT ATG ATC CTT GAC GTT GAT TAC (where NdeI site in bold and start codon underlined) and GGG AAA GGA TCC TCA CTT CTT CTT CCC CTT C (where BamHI site shown in bold type). The PCR products were digested, purified, and ligated into a high expression level vector using standard methods. Plasmid clones were transformed into BL21(DE3). Recombinant bacterial clones were grown using standard procedures and JDF-3 polymerase mutants were expressed in the absence of induction. The exonuclease and polymerase activities of recombinant clones were assayed using bacterial lysates. Typically, crude extracts were heated at 70° C. for 15-30 minutes and then centrifuged to obtain a cleared lysate.
There are several methods of measuring 3′ to 5′ exonuclease activity known in the art, including that of Kong et al. (Kong et al., 1993, J. Biol. Chem. 268: 1965) and that of Southworth et al. (Southworth et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 5281), the full contents of both of which are hereby incorporated by reference. The exonuclease activity of wild type and active mutant polymerases as measured by the Kong et al. method were as follows;
Exo Activity (U/mg):
The combination exonuclease mutant D141A+E143A was made as described in section L.
The E143A JDF-3 mutant (clone #550) exhibited significantly reduced 3′-5′ exo activity and was chosen for further mutagenesis to improve incorporation of ddNTP and other nucleotide analogs. Other JDF-3 mutants with substantially reduced exonuclease activity could have been used for this purpose, such as the JDF-3 D141T mutant. for experiment or applications requiring the absolute elimination of 3′ to 5′ exonuclease activity, the double mutant D141A+E143A was preferred.
D. Error-Prone PCR Amplification of the JDF-3 DNA Polymerase Gene
Random mutations were introduced into exo− JDF-3 by amplifying the entire gene (clone #550) under conditions which did not support high fidelity replication. To broaden the spectrum of potential mutations, three different PCR enzymes were used under error-prone conditions.
In the preferred mode, ten reactions of 100 μl each were amplified with each PCR enzyme.
i. Amplification with Taq DNA polymerase:
Reaction Mixture
Cycling Parameters
PCRs were carried out using Stratagene's ROBOCYCLER™40 Temperature Cycler with a Hot Top assembly. The following cycling conditions were used:
Cycling Parameters
PCRs were carried out using Stratagene's ROBOCYCLER™40 Temperature Cycler with a Hot Top assembly. The following cycling conditions were used:
Cycling Parameters
PCRs were carried out using Perkin-Elmer's 9600 Temperature Cycler. The following cycling conditions were used:
Earlier versions of the mutant libraries were made with the forward primer 461, which contains an EcoR I site. When products amplified with primers 461 and 923 were restriction digested and cloned into the lambda vector as described in the following section, JDF-3 DNA polymerase was synthesized as a fusion protein with the first 39 amino acids of the vector-encoded β-galactosidase (lacZ) protein.
The clones isolated using primer 461 were designed as p#.
The preferred mode of amplification and cloning utilizes the forward primer 721, which also contains an EcoR I site followed by three consecutive in-frame stop codons and a ribosome binding site. This arrangement allows the JDF-3 DNA polymerase to be translated without any vector-derived residues at the amino terminus. The clones isolated from libraries constructed with the forward primer 721 were designated as 1-# to differentiate them from the p# series of clones.
E. Preparing PCR Products for Cloning
PCR products were purified and concentrated with the STRATAPREP™ PCR Purification kit (Stratagene catalog number 400771). The PCR products were then digested with 50 units of Xho I and 50 units of EcoR I in 1.5× Universal buffer (10× Universal Buffer: 1M KOAc, 250 mM Tris-Acetate (pH 7.6), 100 mM MgOAc, 5 mM β-mercaptoethanol and 100 μg/ml BSA) for one hour at 37° C. The digested samples were run on a 1% agarose, 1×TBE gel and visualized with ethidium bromide staining. The 2.3 kb amplification product was gel isolated and purified with the STRATAPREP™ DNA Gel Extraction Kit (Stratagene catalog number 400766).
F. Cloning PCR Inserts into the Uni-Zap®XR Lambda Vector
200 ng of purified amplification product was ligated with 1 μg of UNI-ZAP®XR Lambda Vector (Stratagene catalog #239213), which had been predigested with EcoR I and ho I and then dephosphorylated with alkaline phosphatase (Stratagene catalog number 237211). The DNAs were ligated using 2 units of T4 DNA ligase (Stratagene catalog number 600011) and 0.5 mM ATP in 1× ligase buffer (50 mM Tris-HCL (pH 7.5), 7 mM MgCl2, 1 mM DTT) in reaction volumes of 10 to 15 μl. Ligations were carried out at 16° C. for a minimum of 16 hours.
G. Lambda Packaging and Bacterial Infection
Two microliters of each ligation reaction were packaged with GIGAPACK® III Gold Packaging extract (Stratagene catalog #200201) for 90 minutes at room temperature before being stopped with 500 μl SM buffer (50 mM Tris pH 7.5, 100 mM NaCl, 8 mM MgSO4 and 0.01% gelatin) and 20 μl of chloroform. The packaged lambda vectors were plated on E. coli XL1-Blue MRF′ host cells.
H. Dideoxynucleotide Screening
Mutant polymerase libraries were screened using a variation of the technique used by Sagner et al (Sagner, G., Ruger, R., and Kessler, C. (1991) Gene 97:119-123). Lambda phage clones were plated at a density of 10-20 plaques per square centimeter. Proteins present in the plaques were transferred to filters and moistened with polymerase screening buffer (50 mM Tris (pH 8.0), 7 mM MgCl2, 3 mM β-ME). The filters were kept between layers of plastic wrap and glass while the host cell proteins were heat-inactivated by incubation at 65° C. for 30 minutes. The heat-treated filters were transferred to fresh plastic wrap and approximately 35 μl of the polymerase assay cocktail was added for every square centimeter of filter. Polymerase assay cocktail consisted of 1× cloned Pfu magnesium-free buffer (Stratagene catalog #200534), 125 ng/ml activated calf thymus or salmon sperm DNA, 1.29 μCi/ml α-33P ddNTP (Amersham), and 0.5 mM MnCl2. Initial screening was done in the presence of MnCl2, but the preferred method was to screen in 1×Taq Polymerase buffer (1.5 mM MgCl2). The filters were sandwiched between plastic wrap and glass again and incubated at 65° C. for one hour, and then at 70° C. for one hour and 15 minutes. The filters were washed three times in 2×SSC for five minutes each time before being rinsed twice in 100% ethanol and dried on a vacuum dryer. The filters were exposed to X-ray film for approximately 16 hours. Plaques corresponding to strong signals were cored and placed in SM buffer. The positive primary plaques were replated at more dilute concentrations and assayed under essentially similar conditions to allow the purification of isolated plaques.
Dye-Dideoxynucleotide Screening
To detect mutant polymerases with improved capacity for dye-deoxynucleotide and dye-dideoxynucleotide utilization, the JDF-3 mutant DNA polymerase library was screened as described previously with the following exceptions:
Polymerase assay cocktail for Flu-12-dUTP screening:
0.9×Taq Buffer (Stratagene Catalog #200435), 65 μM dATP, 65 μM dCTP, 65 μM dGTP, 65 μMdTTP, 0.3 μM Fluoresceince-12-dUTP (Stratagene in-house production), 0.75 μg/μl activated calf thymus DNA.
Polymerase Assay Cocktail for ROX ddNTP
1×Taq Buffer, 0.9 μM dATP, 0.9 μM dCTP, 0.9 μM dGTP, 0.9 μl TTP, 0.6 μM ROX ddATP (New England Nuclear (NEN) NEN478), 0.06 μM ROX ddGTP (NEN NEL479), 0.06 μM ROX ddCTP (NEN NEL477), 0.06 μM ROX ddUTP (NEN NEL476), 0.84 μg/μl activated calf thymus DNA. (Note: A screening system without ROX ddGTP is the preferred method since DNA polymerases do not discriminate against ddGTP).
Polymerase Assay Cocktail for Fluroesceine ddUTP
1×Taq Buffer, 70 μM dATP, 70 μM dTTP, 70 μM dCTP, 15 μM dTTP, 1 μM Fluroesceine-12-ddUTP (NEN NEL401), 0.84 μg/μl activated calf thymus DNA.
Antibody Binding to Fluroesceine
The filters were blocked overnight with 1% non-fat dry milk dissolved in TBST (50 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20) at 4° C. The filters were washed briefly in TBST before alkaline phosphatase conjugated anti-fluoresceine antibody from the Illuminator kit (Stratagene catalog #300360) was added at a 1/10,000 dilution in 50 ml TBST. The antibody was detected with NBT/BCIP at concentrations of 0.3 mg/ml and 0.15 mg/ml respectively in a buffer composed of 100 mM Tris pH 9.5, 100 mM NaCl, and 5 mM MgCl2.
Antibody Binding to Rhodamine
Anti-ROX antibody (Zymed cat. no. 71-3600 rabbit Rhodamine (5-ROX polyclonal, 1 mg/ml)) was diluted to 1:1000 in TBST. The blocked filters were blotted briefly to remove excess moisture then laid on plastic wrap and covered with 2.5 ml of the diluted antibody solution. An additional sheet of plastic wrap was laid over the filters before incubation at room temperature for 1 hour. The filters were washed briefly three times with TBST, then washed three times with gentle agitation for 15 minutes each time. The washed filters were incubated with alkaline phosphatase conjugated goat anti-rabbit antibodies diluted 1:5000 in TBST. The filters were incubated with the antibody for one hour then detected with NBT/BCIP as described previously.
I. Dideoxynuclcotide Qualification
Lambda phage clones which incorporated 33P-labeled ddNTPs in the primary library screen were re-screened to verify polymerase activity and to assess the contribution of the divalent metal ion to 33P-ddNTP incorporation. The clones selected during this round of screening were designated as p#. These clones all contained an amino-terminal tag, as discussed in the section entitled “Forward Primers”.
Eighteen mutants were chosen for evaluation. One microliter of phage isolated from each purified plaque was placed on each of three E. coli XLI-Blue MRF′ lawns. Phage containing a parental copy of exo− JDF3 DNA (#550 clone) were also spotted on the grid. The plaques formed by the phage were transferred to filters and treated as described in the preceding screening section with the exception of the final buffer composition. The buffers used for each filter (filters 1-3) are as follows:
Filter 1: Dideoxynucleotide Screen with Manganese Chloride
33P ddNTPs
Filter 2: Dideoxynucleotide Screen with Magnesium Chloride
33P ddNTP
Filter 3: Deoxynucleotide Screen with Magnesium Chloride
Results are shown in
Dye-Dideoxynucleotide Qualification
As described in the previous segments, primary lambda clones were spotted on an E. coli lawn and re-screened with the appropriate antibody or antibodies.
J. Excision of Lambda Clones
When incubated with helper phage under suitable conditions, Lambda Zap™ vectors are designed to produce phagemid copies of the part of the vector containing pBluescript (SK−) and the insert. This process yields a plasmid (pBluescript SK−) vector carrying the same insert that was contained in the lambda clone. Excision of clones with the desired phenotype was carried out according to the instructions in the EXASSIST™ system (Stratagene catalog #200253).
K. Sequence Analysis of Mutants
The mutants were sequenced by Sequetech Corporation (Mountain View, Calif.) using the following primers:
L. Dideoxynucleotide Mutagenesis
To conclusively identify the amino acids contributing to reduced ddNTP discrimination, individual point mutations were introduced into the exo− JDF-3 #550 clone using the QUIKCHANGE™ Site-Directed Mutagenesis Kit (Stratagene catalog #200518). The following mutants were prepared: L408H, L408F, P410L, A485T, S345P, D373Y, A619V, and L631V. In addition, a double mutant (P410L/A485T) was constructed by introducing the A485T mutation into the exo− JDF-3 P410L mutant clone. To completely eliminate all 3′ to 5′ exonuclease activity, the mutation D141A was added to all clones. A pre-existing 5′ to 3′ exonuclease mutation (E143A) was present in the parental template JDF-3 550.
Dye-Dideoxynucleotide Mutagenesis
To conclusively identify amino acids responsible for contributing to reduced discrimination of dye nucleotides, the mutation S345P was generated alone and in combination with the P410L and P410L+A485T.
M. Preparation of Heat-Treated Bacterial Extracts
E. coli SOLR cells containing the excised plasmid were grown overnight at 37° C. The cells contained in 500 μl of culture were collected by microcentrifugation. The cell pellets were resuspended in 50 μl of 50 mM Tris (pH 8.0). Lysozyme was added to a final concentration of 1 μg/μl, and the cells were lysed during a 10 minute incubation at 37° C., followed by 10 minutes at 65° C. The heat-inactivated cell material was collected by microcentrifugation and the supernatants were assayed for dNTP and ddNTP incorporation as described below.
N. Purification of JDF-3 and JDF-3 polymerase mutants
One method for purifying exo− JDF-3 DNA polymerase involves ammonium sulfate fractionation, followed by Q Sepharose and DNA cellulose columns. A second method has been developed to allow rapid purification of JDF-3 polymerase mutants, and entails adsorption of contaminants on a HiTrap Q column, followed by gradient elution from a HiTrap heparin column (section iii).
i. Preparation of Bacterial Lysate.
Frozen cell paste (3-14 grams) was resuspended with 3× volume of lysis buffer, consisting of 50mM Tris-HCl (pH 8.0), 1 mM EDTA, and 10 mM β-mercaptoethanol. Lysozyme was added to 0.2 mg/ml and PMSF was added to 1 mM final concentration. The cells were lysed on ice over a period of 1 hour. The lysate was then sonicated for 2 minutes (90% duty, level of 2×2.5, 1×3.0). Following sonication, the lysate was heated at 65° C. for 15 minutes to denature bacterial proteins. The heated lysate was then centrifuged for 30 minutes at 14.5K rpm in a Sorvall RC-2B centrifuge using a Sorvall SS-34 rotor, and the supernatant was recovered.
ii. Ammonium Sulfate Fractionation and Q Sepharose/DNA Cellulose Chromatography (Method 1)
Ammonium sulfate was added to the bacterial lysate to a final concentration of 45%. The ammonium sulfate was added over a period of 15 minutes, and the mixture was stirred for an additional 30 minutes. The mixture was centrifuged as described above, and the supernatant was recovered. Additional ammonium sulfate was then added to bring the final concentration to 65%. The mixture was centrifuged as described above, and the supernatant removed. The pellet was resuspended in 10 ml of buffer A consisting of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM β-mercaptoethanol, 0.1% (v/v) Tween 20, and 10% (v/v) glycerol. The supernatant was dialyzed overnight against 2 changes of buffer A (3 liters each).
The dialysate was loaded onto a 2.6×9.4 cm Q-Sepharose Fast Flow column (50 mls), pre-equilibrated in buffer A. The column was washed with buffer A until the absorbence (OD280) approached baseline. The column was then eluted with a gradient from 0 to 1 M NaCl/buffer A. Fractions were collected, and analyzed by SDS-PAGE and DNA polymerase activity assays (see below). Active protein typically eluted between 130 and 240 mM NaCl. Active fractions were pooled and dialyzed overnight against 2 changes of buffer B (3 liters each), consisting of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM β-mercaptoethanol, 0.1% (v/v) Tween 20, 10%(v/v)glycerol, and 50 mM NaCl.
The Q-Sepharose eluate was then loaded onto a 1.6×4.9 cm (10 mls) DNA cellulose column, equilibrated in buffer B. The column was washed with buffer B until the absorbence (OD280) approached baseline. The column was then eluted with a gradient from 50 to 1000 mM NaCl/buffer A. Fractions were collected, and analyzed by SDS-PAGE and DNA polymerase activity assays. Active protein typically eluted between 280 and 360 mM NaCl. Active fractions were pooled and dialyzed overnight against JDF-3 final dialysis buffer, consisting of 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% (v/v) Tween 20, 0.1% (v/v) Igepal 630, 10 μg/ml BSA, and 50% (v/v) glycerol.
iii. HiTrap Q/HiTrap Heparin Chromatography (Method 2)
The preferable method for rapid purification of multiple mutants is as follows. Bacterial cell lysates were prepared as described for method 1, except that Tween 20 and Igepal CA 630 were added to a final concentration of 0.01% (v/v) just prior to the heat denaturation step, and a heat denaturation temperature of 72° C. was used.
The lysate was loaded onto a 1.6×2.5 cm (5 mls) HiTrap Q column (pre-packed column from Pharmacia), pre-equilibrated in buffer C consisting of 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, 10 mM β-mercaptoethanol, 0.1% (v/v) Tween 20, and 0.1% (v/v) Igepal CA 630. The column was washed with buffer C until the absorbence (OD280) approached baseline. The flow through fractions (OD280 absorbence above background) were collected and then loaded onto a 1.6×2.5 cm (5 mls) HiTrap heparin column (pre-packed column from Pharmacia), pre-equilibrated in buffer D consisting of 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, 1 mM DTT, 0.1% (v/v) Tween 20, 0.1% (v/v) Igepal CA 630, and 10% glycerol (v/v). The column was washed with buffer D until the absorbence (OD280) approached baseline. The column was then eluted with a gradient from 0 to 1 M KCl/buffer D. Fractions were collected, and analyzed by SDS-PAGE and DNA polymerase activity assays. Active protein typically eluted between 390 and 560 mM NaCl. Active fractions were pooled and dialyzed overnight against JDF-3 final dialysis buffer (see above). Purified polymerases were stored at −20 C.
iv. Analysis of Purified Proteins
The concentrations of JDF-3 and mutant DNA polymerases were determined relative to a BSA standard (Pierce), using Pierce's Coumassie Blue Protein assay reagent. In addition, the purity and relative protein concentrations of different polymerase preparations were verified by SDS-PAGE. Polymerase samples were electrophoresed on 4-20% Tris-glycine gels (Novex), and the gels were silver-stained using standard procedures.
O. Nucleotide Incorporation Assay
DNA polymerase activity was measured using purified JDF-3 polymerase mutants or heat-treated bacterial extracts prepared from various mutant clones. DNA polymerase activity was measured by monitoring the incorporation of 3H-TTP into activated calf thymus DNA. A typical DNA polymerase reaction cocktail contained:
Incorporation was measured by adding 1 μl of polymerase samples to 10 μl aliquots of polymerase cocktail. DNA polymerase samples were diluted in a suitable storage buffer (e.g., 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% (v/v) Tween 20, 0.1% (v/v) Igepal 630, 10 μg/ml BSA, and 50% (v/v) glycerol). Polymerization reactions were conducted for 30 minutes at 72° C. Extension reactions were quenched on ice, and then 5 μl aliquots were spotted immediately onto DE81 ion-exchange filters (2.3 cm; Whatman #3658323). Unincorporated [3H]TTP was removed by 6 washes with 2×SCC (0.3 M NaCl, 30 mM sodium citrate, pH 7.0), followed by a brief wash with 100% ethanol. Incorporated radioactivity was measured by scintillation counting. Reactions that lacked enzyme were also set up along with sample incubations to determine “total cpms” (omit filter wash steps) and “minimum cpms” (wash filters as above).
Cpms bound is proportional to amount of polymerase activity present per volume of bacterial extract. The volume of bacterial extract (0.25-1 μl) which brought about incorporation of approximately 10,000 cpms was determined for use in subsequent nucleotide analog incorporation testing.
P. Quantitating ddNTP Incorporation Efficiency
JDF-3 polymerase mutants were evaluated to assess relative ddNTP incorporation efficiency. Nucleotide incorporation was measured in the presence of varying concentrations of each ddNTP terminator (ddATP, ddCTP, ddGTP, and ddTTP). Since ddNTP incorporation produces non-extendable termini, polymerization is strongly inhibited for polymerases that incorporate ddNTPs efficiently. Comparisons of ddNTP concentrations that bring about 50% inhibition of nucleotide incorporation (I50%) can be used to compare ddNTP incorporation efficiency of different polymerases or polymerase mutants. Comparisons of I50% values for ddATP, ddCTP, ddGTP, and ddTTP can be used to identify mutants with reduced selectivity for particular bases. Such mutants would be expected to produce more uniform DNA sequencing ladders.
To measure incorporation of individual ddNTPs, cocktails were prepared which consisted of varying concentrations of the ddNTP of interest, and a total of 200 μM of each nucleotide triphosphate. For example, the incorporation of ddATP by wild type JDF-3 polymerase was measured at 0, 40, 80, 120 and 160 μM ddATP. In these reactions, dATP concentrations were adjusted to 200, 160, 120, 80, and 40 μM, respectively, so that the total amount of adenine nucleotide triphosphate was 200 μM. In comparison, mutants were assayed using ddATP concentrations of 0, 5, 10, and 20 μM ddATP, and adjusted dATP concentrations of 200, 195, 190, and 180 μM, respectively (dATP+ddATP=200 μM). Additional cocktails were prepared to measure ddCTP, ddGTP, and ddTTP incorporation. To assess ddNTP incorporation by JDF-3 mutants at 3 different ddNTP concentrations, 12 reaction cocktails were prepared consisting of:
To each of 12 reaction cocktails was added the appropriate amounts of dNTPs and ddNTPs as summarized below:
Incorporation was measured by adding 1 μl of appropriately diluted bacterial extract (10,000 cpms) to 10 μl of each polymerase cocktail. Polymerization reactions were conducted for 30 minutes at 72° C. The extension reactions were counted as described above.
Reactions that lacked enzyme were also set up along with sample incubations to determine “minimum cpms” (wash filters as above). To determine % activity as a function of ddNTP concentration, background (“minimum cpms” value) was first subtracted from each of the sample cpms. “Total cpms”, which are equivalent to 100% activity (0 ddNTPs), are determined by averaging the corrected cpms for the 4 reactions lacking ddNTPs (A-0, G-0, C-0, and T-0). Percent remaining activity was then calculated by dividing corrected sample cpms (with ddNTPs) by the corrected total cpms (average 0 ddNTPs).
Percent activity was plotted as a function of ddNTP concentration. I50% values for each ddNTP (ddNTP concentration which inhibits nucleotide incorporation by 50%) were determined for each mutant. Comparisons allowed the identification of mutants with improved ddNTP incorporation relative to wild type JDF-3.
Initial studies used purified enzymes, and I50% values were determined from inhibition plots employing 40-160 μM ddNTPs. The results in Table V show that mutants p8 (P410L), p11 (P410L), and p 12(A485T) are inhibited by lower concentrations of ddNTPs than the parental exo JDF-3 polymerase. Greater sensitivity indicates that the mutants incorporate all four ddNTPs more efficiently than the original JDF-3 polymerase.
For enzymes which preferentially incorporate TTP over ddTTP (exo− JDF-3, exo− Pfu), the use of increasingly higher concentrations of ddTTP (80-160 μM) and correspondingly lower concentrations of TTP (115-35 μM), in combination with a constant amount of [3H]TTP (5 μM), leads to an increase in cpms incorporated with increasing ddNTP concentration. Therefore, in these initial experiments (where ddTTP>120 μM), I50% values for TTP are artificially high. While they can be used to compare ddTTP incorporation among different polymerase mutants, they can not be used to assess reduced/enhanced preference for ddTTP relative to ddCTP, ddGTP, or ddATP.
To allow a larger number of mutant clones to be screened, subsequent experiments employed bacterial extracts containing JDF-3 polymerase mutants. In addition, sensitivity was improved by using lower concentrations of each ddNTP inhibitor (5-20 μM). The results in Table VI demonstrate that all of the mutants selected from the primary filter screen exhibited improved incorporation of ddNTPs. Improvements in ddNTP incorporation were as high as >20-fold. All of the mutants containing a mutation at amino acid 408 (L408H/F), 410 (P410L), or 485 (A485T) (referred to as the “primary mutation”) exhibited reduced discrimination against all four ddNTPs. Most, but not all, mutants with the L408H/F primary mutation produced very similar I50% values (<2-fold difference) for all four ddNTPs, indicating that base selectivity is diminished or absent.
Q. Sequencing with Purified JDF-3 Polymerase Mutants
i. Sequencing with Radioactively Labeled Dideoxynucleotides
1 to 2 μl of purified enzyme was substituted into the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham-Pharmacia #US79750). The samples were processed according to the manufacturer's instructions using the control primer and template provided with the kit. Three microliters of each sequencing reaction were loaded onto a 6% acylamide-7M urea, 1×TBE CASTAWAY™ Precast gel (Stratagene catalog #s 401090 and 401094). When the bromophenol blue indicator dye reached the end of the gel, the gel was fixed, dried and exposed to film for 24-72 hours (
The results in
ii. Sequencing with a Radioactively Labeled Primer and Fluorescent Dideoxynucleotides
Different DNA polymerases and polymerase mutants will exhibit varying degrees of discrimination against the dye moieties on the dideoxynucleotide analogs. An assessment of usage of dye-labeled dideoxynucleotide analogs by the JDF-3 polymerase mutants was carried out. The procedure used was as follows:
a. Primer Labeling
The sequencing primer SK was radioactively labeled with the KINACE-II™ Kinasing Kit (Stratagene catalog #200390). The incubation reaction (40 μl) contained the following components:
The reaction was incubated at 37° C. for 45 minutes. The primer was purified away from free nucleotides with a size exclusion matrix (NUC TRAP® Stratagene catalog number 400701).
b. Dye Labeled-Dideoxynucleotide: DNTP Ratios
Fluorescent dideoxynucleotides were purchased from New England Nuclear (NEN):
Incorporation was measured using 3 different concentrations of dye labeled
c. Preparation of the DNA Sequencing Reaction Mixtures
Four polymerases were tested for utilization of dye-labeled ddNTPs, exo− JDF-3 (#550 clone), Thermo Sequenase (4u/μl), JDF-3 P410L (clone p8 with ancillary mutations and an amino-terminal tag) and JDF-3 L408H (clone 1-1). A mixture containing the following reagents was assembled:
10× Reaction Buffer 1
10×1.5 mM MgCl2 Buffer
2.5 μl of each dye-labeled ddNTP terminator (ddGTP, ddATP, ddTTP and ddGTP was aliquotted separately into one of four tubes. 4.5 μl of each polymerase reaction was added to each of the four tubes, to give a final reaction volume of 7 μl.
d. Cycle Sequencing Reactions
The samples were cycled in a RoboCycler®96 Temperature Cycler with a Hot Top Assembly (Stratagene Catalog #400870 and #400894) using the following conditions:
4 μl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) was added to each of the amplified reactions before heating them to 99° C. for five minutes. The samples were electrophoresed on a 6% CASTAWAY™ gel as described above. The gels were dried and then exposed to film for 72 hours (
iii. Sequencing with Double-Mutant exo− JDF-3 DNA polymerase.
To verify that changes at residues 408, 410, and 485 were sufficient to improve ddNTP incorporation, individual mutations were introduced into the parental 550 (JDF-3 exo− DNA polymerase) clone by site-directed mutagenesis. In addition, point mutations were combined to examine whether they resulted in further improvements in dideoxynucleotide incorporation over polymerases bearing single mutations.
DNA sequencing reactions consisting of 1× reaction buffer, 0.15 pmol/μl long −20 primer, and 10 ng/μg pBluescript KS were prepared as follows:
18 μl of the cocktail listed above was aliquotted into the appropriate number of tubes (one per polymerase). Each polymerase (2 μl) was added to an aliquot of cocktail and the tubes were mixed well. Each resulting polymerase mixture (4.5 μl) was then added to each of four tubes, already containing 0.06 mM of one of the four −33P-dideoxynucleotides (ddATP, ddTTP, ddGTP or ddTTP; 1500Ci/mmol; 450 μCi/ml) and 6 mM each deoxynucleotide in a volume of 2.5 μl.
The sequencing reactions were cycled in a ROBOCYCLER®96 temperature cycler with a Hot Top Assembly using the following conditions:
Stop solution (μl; 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) was added to each reaction before heating to 99° C. for five minutes. Each sample (4 μl) was loaded onto a 6% acrylamide denaturing CastAway gel. The gel was run and treated as described previously.
iv. Ribonucleotide Incorporation by JDF-3 Polymerase Mutants.
A primer annealed to single stranded DNA template was extended in a mixture containing all ribonucleotides or all deoxynucleotides with the mutant and progenitor polymerases.
M13mp18+ single stranded DNA was annealed to 95× molar excess of the 38mer primer by heating the mixture to 95° C. and cooling slowly at room temperature.
38mer primer: 5′ GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT 3′
Preliminary assays were carried out to determine what dilutions of enzyme would be necessary to examine the incorporation activity at non-maximal levels. The final assay solutions were composed as described below:
Ribonucleotide Mixture
Deoxyribonucleotide Mixture
Nine microliters of the polymerase-free mixtures were placed in 0.2 ml tubes before the polymerases were added. The samples were incubated at 72° C. in a ROBOCYCLER®96 temperature cycler with Hot Top Assembly (Stratagene Catalog Nos. 400870 and 400894). The deoxyribonucleotide mixture was removed at 2 minutes and placed at approximately 2° C. The ribonucleotide mixture was incubated for 30 minutes. Seven microliters of the assay mixture were spotted onto DE81 filter circles (Whatmann) and dried prior to being washed three times in 2×SSC (0.3M NaCl, 0.03M sodium citrate) for five minutes each wash. The filters were rinsed twice in ethanol and allowed to dry before being quantified with a scintillation counter.
Background counts per minute (CPM) for the deoxyribonucleotide and the ribonucleotide reactions were subtracted from the respective averaged CPM value of duplicate samples for each enzyme. The background-corrected ribonucleotide CPM value was divided by the background-corrected deoxyribonucleotide CPM value (
v. Ribonucleotide Sequencing with JDF-3 Polymerase Mutants.
Ribonucleotides incorporated into a deoxyribonucleotide polymer are susceptible to alkali hydrolysis which can produce a sub-population of polymer lengths. When labeled primer is extended in the presence of a particular ribonucleotide base (for example ATP) and the four deoxyribonucleotide bases, the fragments resulting from alkali hydrolysis create a population of different lengths, which correspond to all the possible positions where ATP was incorporated. When those fragments are size separated, their migration pattern, with respect to other ribonucleotide base (CTP, UTP and GTP) hydrolysis products allows the template sequence to be read. As described previously, most DNA polymerases discriminate against non-conventional deoxynucleotides. A subset of the JDF-3 DNA polymerase mutants which allow improved uptake of the unconventional dideoxynucleotides also show improved tolerance for ribonucleotide incorporation.
100 ng of the 38mer primer was kinased with γ-33P according to the instructions in the KINACE-IT™ Kinasing Kit (Stratagene catalog #300390).
38mer primer: 5′ GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT 3′
The labeled oligonuclcotide was purified from contaminating free nucleotides with a NUC TRAP® Probe Purification Column (Statagene catalog #400701) in 10T.1E (10 mM Tris pH 8.0, 0.1 mM EDTA). Labeled oligonucleotide (˜7 picomoles) was annealed to 0.09 pmoles M13 mp18+ by heating to 95° C. then cooling to room temperature in the presence of 0.32 mM MgCl2.
Extension Components
Eight microliters of a cocktail containing the first three components listed above were aliquoted into a 0.2 ml tube. 1 μl of polymerase and 1 μl of 2 mM, 0.2 mM or 0.4 mM ATP were added and the reaction was incubated at 72° C. for 15 minutes. The reaction volume was brought to 100 μl with 1×cPfu polymerase buffer and transferred to a 1.5 ml tube. After heating the reactions in the presence of 70mM NaOH for 15 minutes at 100° C., the reaction was neutralized with 70 mM HCl and precipitated through the addition of 10 μl 3M sodium acetate and 327.51 μl of ethanol. The samples were microcentrifuged for 30 minutes at 14 krpm before the supernatant was removed and the pellet washed in 80% ethanol. After vacuum drying, the samples were resuspended in 5 μl of sequencing stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) and 2.51 μl was loaded on a 6% acylamide-7M urea, 1×TBE CASTAWAY™ Precast gel (Stratagene catalog numbers 401090 and 401094). The gels were run at 50 watts until the bromophenol blue dye migrated past the bottom of the gel after which the gel was fixed, dried and exposed to film for 72 hours.
Sequencing ladders for JDF-3 550 (wild-type nucleotide incorporation) and all the mutants tested were visible at the 200 μM and 20 μM ATP level. At the 4 μM level, only the L408H and L408F mutants produced ladders (data not shown).
vi. Sequencing with Dye-Dideoxynucleotide Terminators
Primer was extended in the presence of FAM ddCTP (NENNEL481). The sequence reactions were purified and run on an ABI 370.
Reaction conditions for cycle-sequencing were as described below:
1×cPFU buffer, 200 ng pBluescript II KS plasmid, 3 pmole T7 primer, 0.23 mM dCTP, 0.23 mM dATP, 0.23 mM dTTP, 0.23 mM dGTP with 0.046 mM FAM ddCTP. The samples were cycled in a Perkin-Elmer cycler in 10 μl volumes for 25 cycles of the temperatures and times described below:
The samples were purified using CentriSep columns according to the manufacturer's instructions. After drying, the samples were resuspended in 3 μl of a loading dye comprised of 66.7% deionized formamide, 16.7 mg/ml Blue Dextran, and 8.3 mM EDTA. Samples were heated at 95° C. for three minutes and loaded on a 5% LongRangen gel in an ABI PRISM 377 DNA sequencer.
Data was processed in Gene Scan 2.1.
Labeling of DNA.
The modified DNA polymerases of the invention are applicable to labeling of DNA. It is known to those skilled in the art that there are several means by which to label DNA, including the incorporation of radiolabeled nucleotides. One such common means is by random priming, which enables one of skill in the art to generate labeled DNA fragments, typically about 50 to about 1000 bases long. The procedure described herein are adapted from F. Ausubel et al., Short Protocols in Molecular Biology, Third Edition, John Wiley and Sons, Inc., 1995.
As a first step toward random priming DNA, a reaction mix containing 2.5 microliters 0.5 mM 3dNTP (dCTP, dGTP, TTP, each at 0.5 mM), 50 μCi[−32P]dATP, 1 microliter of 3 to 8 units/microliter DNA polymerase in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 0.05 mg/ml bovine serum albumin is prepared in a total volume of 11 microliters and incubated on ice. Next, about 30 to about 100 ng of DNA is mixed with about with 1 to 5 μg of random hexanucleotides in 14 microliters and boiled for 2 to 3 minutes and then placed on ice. The 11 microliter reaction mix is then added to the DNA/random hexamer mix, and the random priming reaction is incubated over 10 minutes to as much as 4 hours at room temperature. To stop the reaction, 1 microliter 0.5 M EDTA, 3 microliters 110 mg/ml tRNA, and 100 microliters 10 mM Tris-HCl, pH 7.4 is added and the mixture is extracted with phenol. The labeled DNA is then separated from unincorporated radioactive precursors by chromatography.
R. Gel Assay for Dye-Dideoxynucleotide Incorporation
A labeled oligonucleotide duplex was extended with a mixture of dideoxynucleotides and dye-dideoxynucleotides. When the duplex was separated on a denaturing 20% Acrylamide/7 M urea gel, labeled oligonucleotides terminated with a dideoxynucleotide could be resolved from oligonucleotides terminated with dye-deoxynucleotides.
Oligonucleotides:
32P-TAACGTTGGGGGGGGGCA→
The 5′ end of 259C was labeled and purified as described in Section Q.ii.a except that 32Pγ-ATP was used. The labeled oligonucleotide 259C was at a concentration of approximately 0.7 ng/μl. The complimentary oligonucleotide (258C) was added as an equal concentration, heated to 95° C. for three minutes, 50° C. for 5 minutes and room temperature for 20 minutes. Heat killed lysates of the relevant mutants were prepared as described in Example section C. The reactions were incubated in a 5 μl volume composed of 30 mM Tris pH 8.0 and 3 mM MgCl2 with a nucleotide mixture totaling 0.1 mM. The ratio of ddTTP to FLU ddUTP or ROXddUTP was 10:1. The dimer was present at a concentration of 1.2 picomoles and 0.5 μl of enzyme or crude lysate or purified enzyme was added to the reaction before incubation at 50° C. in the RobeCycler® Gradient 96 Temperature Cycler with Hot Top. The samples were incubated for 20 s before 3 μl of a formamide based loading dye was added and the samples were heat-denatured at 95° C. for 3 minutes then loaded onto a 20% acrylamide/7 M urea gel and subjected to electrophoresis at a constant 60 watts. The gel was exposed to X-ray film and the film was analyzed in the EagleEye® Eagle Sight software package.
The modified DNA polymerases of the invention are also applicable to identify a nucleotide at a given position of a template DNA molecule, i.e., by mini-sequencing. For example, the JDF-3 DNA polymerase P410L/A485T mutant (JDF-3 D141A/E143A/P410L/A485T) generates the longest and most uniform radioactive DNA sequencing ladders using low ddNTP/dNTP ratios (1/100), indicating efficient ddNTP incorporation, minimal base selectivity, and high polymerase activity. This example describe the properties of this JDF-3 DNA polymerase P410L/A485T mutant and a procedure for optimizing the conditions for mini-sequencing using the mutant polymerase.
A. Experimental Protocol
i. Materials
StrataPrep PCR columns, StrataPrep DNA gel extraction columns, cold ddNTPs, calf alkaline phosphatase and pBluescript II were from Stratagene. Rhodamine labeled-ddNTPs were purchased from NEN. EDTA/blue dextran, rhodamine dye-matrix standards, and the SNaPshot ddNTP primer extension kit were purchased from Applied Biosystems. ThermoSequenase (Taq F667Y mutant) was from Amersham Pharmacia Biotech. Long Ranger polyacrylamide gels (6%) were purchased from BMA. Shrimp alkaline phosphatase and exonuclease I were from USB corporation. CENTRI-SEP spin columns were purchased from Princeton Separations. Deionized formamide was from Sigma. Oligonucleotides (PAGE purified) whose sequences are listed in Table VII, were purchased from Genset oligos. All other reagents were molecular biology grade.
ii. Primer:Template Formation:
Duplex primer template pairs were formed by annealing the template with 10 fold excess of the appropriate primer in a solution containing 10 mM Tris-HCl (pH 8) and 0.1 mM EDTA using the following temperature regimen: 5 min at 95° C. and then cool slowly to room temperature. Concentrations of the primer:templates are expressed as moles of single stranded templates.
iii. Product Analysis:
The dye labeled products were resolved on 6% polyacrylamide/urea gels and visualized on a Applied Biosystems model 377 DNA sequencer using 3.1.2 GeneScan fragment analysis software for peak identification and fluorescence measurements. A rhodamine dye-matrix was installed on the ABI 377 sequencer according to the manufacturer's protocol.
iv. Purification and Optimal Reaction Buffer of JDF-3 P410L/A485T DNA Polymerase:
JDF-3 P410L/A485T was expressed in XL-10 Gold and purified as described in the “Purification of JDF-3 P410L/A485T mutant” Product Transfer Document by Brad Scott. One unit of enzyme is defined as the amount that will catalyze the incorporation of 10 mmol of total nucleotide into acid insoluble form in 30 minutes at 72° C. The 10× reaction buffer for JDF-3 P410L/A485T contains: 200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4.
v. Enzyme Assays:
a. Kinetic Analysis of Rhodamine Labeled-ddNTP Incorporation and Misincorporation:
Km and Vmax values for rhodamine labeled-ddNTP incorporation were measured by incubating 50 nM primer:template with limiting amounts of polymerase (0.1 units or 5 nM) and varying concentrations of rhodamine labeled-ddNTP ranging from 0.1 nM to 500 nM. Samples were incubated at 60° C. for 10 minutes. The reactions were then quenched with ice-cold 0.2 M EDTA (final concentration). Unincorporated rhodamine labeled ddNTPs were removed by purifying the extended primer:templates on CENTRI-SEP spin columns. Reactions were then dried, and the pellets dissolved in 3:1 formamide:EDTA/blue dextran and analyzed by 6% denaturing PAGE on an ABI 377 sequencer. All peak area quantitations were performed using 3.1.2 GeneScan software and Km and Vmax values were calculated using Lineweaver-Burk plots.
Km and Vmax values for primer:template were measured with limiting amounts of enzyme (0.1 units or 5 nM) in the presence of 100 nM of R110-ddGTP and varying concentrations of primer:template (pBL31G:pBluescript II) ranging from 0.5 to 100 nM. Samples were incubated for 10 minutes at 60° C. The reactions were then quenched, purified from unincorporated rhodamine labeled-ddNTPs, and analyzed as described above.
To determine the kinetics of misinsertion, the steady state Michaelis-Menten Km and Vmax parameters were calculated by incubations of limiting amounts of enzyme (0.1 units or 5 nM) in presence of 50 nM primer:template and varying concentrations of non-complementary rhodamine labeled ddNTP (1 nM to 10,000 nM) for 10 minutes at 60° C. Analysis and quantitations were performed as above.
b. Screen for Fidelity:
Reactions (10 μl) contained 1 unit of enzyme in 1× reaction buffer, 15 nM primer template, 25 nM of unlabeled complementary ddNTP, and 25, 100, 500, or 1000 nM of rhodamine labeled non-complementary ddNTP in four separate reactions. The reactions were incubated in a Perkin-Elmer 9600 for 25 cycles as follows: 96° C. for 10 s, 50° C. for 5 s, and 60° C. for 30 s. The reactions were then quenched with ice-cold 0.2 M EDTA (final concentration) and the products were purified from unincorporated rhodamine labeled ddNTPs and analyzed as described above.
c. Assays for Rhodamine Labeled ddNTP Incorporation:
These experiments were performed using 1 unit of enzyme, 15 nM primer:template (pBL25C and Temp-A, Temp-T, Temp-C, or Temp-G in four separate reactions), and 50 nM dye-ddNTP (TAMRA- or R110-labeled). The reactions were incubated at 60° C. for 10 minutes and then quenched with ice-cold 0.2 M EDTA (final concentration). The products were purified from unincorporated dye-labeled ddNTPs and analyzed as described above.
B. Optimization Procedures
i. Preparation of DNA Templates for Minisequencing:
Fragments containing the SNP(s) of interest are amplified from genomic DNA using standard PCR conditions. In this study, PCR reactions were carried out using 2.5 units of TaqPlus Precision DNA polymerase blend. A 4 kb fragment of the human alpha-1-antitrypsin gene was amplified from 100 ng human genomic DNA using 10 pmol of the pPC26G and pPC29C primers (Table VII). The following program was used in a Robocycler: 1 cycle of 95° C. for 2 min, 30 cycles of 95° C. for 1 min, 58° C. for 1 min, and 72° C. for 4 min, followed by one cycle at 72° for 7 min.
In order to purify the resulting 4 kb fragment from PCR primers and unincorporated dNTPs, the fragment can be: (1) purified using the StrataPrep DNA gel extraction kit; (2) treated with exoI/SAP; or (3) incubated on StrataPrep columns with SAP or CIAP. To treat the PCR fragment with exoI/SAP, 2 units of each enzyme was added to 4 μl of PCR product, and the mixture was incubated for 1 hour at 37° C., followed by 15 minutes at 72° C. to inactivate the enzymes. To purify the PCR amplified fragment using StrataPrep columns, a 50 μl PCR reaction was loaded on a StrataPrep column and processed as described in the StrataPrep column manual, except that before elution, 1 unit of CIAP in 50 μl 1× corresponding reaction buffer was added to the column. The column was incubated at room temperature for 5 minutes, washed, and the PCR fragment was eluted as described in the manual. The eluate was incubated at 72° C. for 15 minutes to inactivate any remaining CIAP. All of these clean up methods produced DNA templates that were pure enough for subsequent minisequencing.
ii. Minisequencing Protocol Using Plasmids or PCR Amplified Fragments:
Minisequencing of pBluescript (0.25 pmol) was carried out using 0.15 pmol of each primer (e.g. pBL25C), 1 unit enzyme, 0.04 μM of R6G-ddA, R110-ddG, and ROX-ddC, and 0.2 μM of TAMRA-ddU. When using PCR amplified fragments, template concentrations as low as 0.02 pmol/rxn were used. All reactions were performed in 10 μl volumes. The thermal cycling program consisted of 25 cycles of 96° C. for 10 s, 50° C. for 5 s, and 60° C. for 30 s in a Perkin-Elmer 9600 or 25 cycles of 96° C. for 50 s, 50° C. for 50 s, and 60° C. for 50 s in a Robocycler.
In order to purify the labeled primers from unincorporated dye-ddNTPs, samples were either treated with SAP or CIAP, or purified using CENTRI-SEP columns according to manufacturers's recommendations. 1 unit of CIAP or 0.5 unit of SAP was added to each 10 μl reaction and incubated at 37° C. for 60 min, followed by 15 minutes at 72° C. to inactivate alkaline phosphatase. Reactions were then dried and pellets were dissolved in 10 μl of 3:1 formamide:EDTA/blue dextran. 1 μl of each reaction was resolved by 6% denaturing PAGE on an ABI 377 sequencer and analyzed using GeneScan 3.1.2 (Applied Biosystems).
C. Results
i. Buffer and Reaction Temperature Optimizations:
Since Thermococcus sp. JDF-3 DNA polymerase is closely related to archaeal P. furiosus DNA polymerase (Pfu), cloned Pfu buffer (10× buffer: 200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X100, and 1 mg/ml BSA) was used as a starting point for buffer optimization. Changes in enzyme activity due to buffer and reaction temperature alterations were determined by measuring R6G-ddATP incorporation using the pBL34A:pBluescript primer:template system. The presence of Triton X-100 and BSA in this buffer was found to create an artifact (double band effect) in sequencing gels (data not shown). Enzyme activity was measured at pH 8.4 and 9.5, in the presence of varying concentrations of KCl (20, 40 or 80 mM), (NH4)2SO4 (5 or 20 mM), and MgSO4 (4 or 8 mM), respectively. None of these changes had a noticable effect on the activity of JDF-3 P410L/A485T. Therefore, cloned Pfu buffer lacking BSA and Triton was identified as the optimal reaction buffer for minisequencing. Furthermore, the activity vs. temperature profile of JDF-3 P410L/A485T showed that nucleotide incorporation did not increase significantly between 60° C. and 72° C. (data not shown). To keep the extension temperature below the melting temperatures of minisequencing primers, all subsequent experiments were performed at 60° C.
ii. Incorporation of Rhodamine-Dideoxyribonucleotides:
Relative incorporation of rhodamine labeled-ddNTPs by the JDF-3 P410L/A485T and Taq F667Y mutants was determined. We used both TAMRA- and R110-labeled ddNTPs, and the amount of incorporated dye-ddNTPs was measured in fluorescense units. These experiments were performed using pBL25C as the primer, and Temp-A, Temp-T, Temp-C, or Temp-G as the complementary template in four separate reactions. The only difference between these four primer:template systems is the SNP site, thereby eliminating the possibility that primer:template sequence has an effect on dye-ddNTP incorporation.
We performed similar rhodamine labeled-ddNTP incorporation experiments using the same number of units of Taq F667Y. As panels A and B in
iii. Kinetic Parameters for Polymerization Reaction:
Km and Vmax values for primer:template and rhodamine-ddNTPs were determined as described in the Experimental Protocol. These values are reported in Table VIII, which compares the kinetic properties of JDF-3 P410L/A485T and Taq F667Y. This comparison establishes that the JDF-3 P410L/A485T and Taq F667 mutants exhibit similar steady-state kinetic parameters, and therefore, have similar affinities for both primer:template and rhodamine-ddNTP substrates.
Furthermore, kinetic parameters in Table VIII were used to determine incorporation efficiency of TAMRA-ddCTP (Vmax/Km=3.3/1=3.3) in comparison to TAMRA-ddATP (Vmax/Km=1.9/0.9=2.1). Incorporation efficiency of TAMRA-ddCTP is 1.5 fold more than TAMRA-ddATP, suggesting that JDF-3 P410L/A485T mutant incorporates different ddNTPs at slightly different rates, confirming the results obtained in
aAll values have ±<30% error and are obtained from at least two independent experiments.
bpBL25C:pBluescript
cpBL34A:pBluescript
dpBL31G:pBluescript
iii. Fidelity:
Fidelity was determined as the tendency of a DNA polymerase to incorporate the correct nucleotide in the presence of increasing amounts of a non-complementary nucleotide. These assays employed constant amounts of primer:template, an unlabeled complementary ddNTP and DNA polymerase, and various concentrations of a rhodamine-labeled non-complementary ddNTP, as described in the Experimental Protocol. The amount of misextended primers is plotted against the ratio of rhodamine-labeled incorrect ddNTP/unlabeled correct ddNTP (
ddC:dA
ddA:dG
ddT:dT
ddC:dC
ddA:dA
ddG:dG
The two mispairs, ddT:dC and ddG:dT, are formed at a significantly higher frequency (3 or 20 fold) by Taq F667Y mutants (ThermoSequenase and AmpliTaq FS) compared to JDF-3 P410L/A485T (
Similar rates of ddG:dT mispair formation were obtained using AmpliTaqFS (Taq F667Y mutant; ABI). AmpliTaqFS is only available in a mixture containing dye-ddNTPs and reaction buffer (SNaPshot kit; ABI). In order to test AmpliTaqFS, we removed dye-ddNTPs by adding 0.5 unit of SAP to 5 μl of the SnaPshot kit mix, followed by incubations at 37° C. for 30 minutes and at 72° C. for 15 minutes. The resulting mix, free of dye-ddNTPs, was then used in fidelity assays.
In order to establish that this difference in fidelity is not sequence specific, we performed similar experiments with two other primer:template systems (pBL25C:Temp-T and pPC34A:4 kb PCR amplified fragment) and obtained similar misincorporation rates for both enzymes (data not shown). We also obtained similar misincorporation rates using TAMRA-labeled ddNTPs as the incorrect ddNTP (data not shown). Therefore, the lower fidelity exhibited by Taq F667Y DNA polymerase is neither sequence specific nor due to increased misincorporation of R110 dyes.
To gain more insight into the mechanism of lower fidelity, we determined the misinsertion frequency for the mispair ddG:dT, which was evaluated in terms of relative Km and Vmax values for the wrong versus correct dye-ddNTP. Efficiency of nucleotide misinsertion was determined opposite a DNA template dT, primed with a 34-nucleotide oligomer (pBL34A). Apparent Michaelis constant (Km) and maximum velocity (Vmax) and relative insertion frequencies were measured for ddATP and ddGTP (Table X).
aAll values have ±<30% error and are obtained from 3 independent experiments.
bI.E., M.E. and M.F. are insertion efficiency, misinsertion efficiency and misinsertion frequency, respectively.
As shown in Table X, the misinsertion frequency of Taq F667Y for ddG:dT is significantly (˜17 fold) higher than that exhibited by JDF-3 P410L/A485T. This difference is mostly due to differences in Km for the wrong dye-ddNTP•primer:template ternary complex. The Taq F667Y mutant exhibits a 7-fold lower Km (higher binding affinity) for wrong nucleotide (ddGTP) opposite dT, compared to the JDF-3 P410L/A485T mutant.
iv. Development of a Minisequencing Kit:
There are five major steps involved in SNP detection by minisequencing and gel electrophoresis: (i) extraction of DNA from blood or tissue samples; (ii) PCR amplification of specific fragments of genomic DNA containing SNPs; (iii) treatment of PCR products prior to minisequencing to remove unreacted PCR primers and dNTPs; (iv) minisequencing of PCR products and purification of extended primers; (v) analysis of fluorescent labeled primers using gel electrophoresis and GeneScan software (ABI). Here, we have optimized steps (iii) and (iv), and developed a minisequencing kit containing enzyme, reaction buffer, rhodamine labeled-ddNTPs, and a control primer:template system.
To optimize step iii, we used three different approaches to purify PCR products from unreacted PCR primers and dNTPs prior to minisequencing as described in the Experimental Protocol. We found that DNA templates purified using StrataPrep columns without CIAP treatment were contaminated with enough residual dNTPs to interfere with subsequent minisequencing. In the presence of trace amounts of contaminating dNTPs, sequencing ladders were produced instead of a single extended primer (
Three different primer:template systems were used to optimize the JDF-3 P410L/A485T minisequencing kit, including pBluescript with primers pBL34A, pBL31G, pBL28T, and pBL25C (Table VII) which will be used as the kit controls (
The concentrations of JDF-3 P410L/A485T, dye-ddNTPs, primer, and template were optimized for minisequencing and fluorescence detection by ABI 377 sequencer (see Experimental protocol for optimized conditions). Higher concentrations of enzyme, dye-ddNTPs, and primer:template will increase the fluorescent signal. However, such increases in signal saturate the ABI 377 detector and result in dye bleed-through (more than one color showing in one spot).
Different rhodamine dye/ddNTP combinations can be used with JDF-3 P410L/A485T (
β-testing of JDF-3 P410L/A485T at Stanford genome technology center was performed using Cy3-ddATP and Cy5-ddCTP, and immobolized primers on Zyomyx aldehyde slides.
The following protocol (Applied Biosystems) should be used in designing primers for minisequencing: (1) Since SNP validation by minisequencing is not flexible with respect to the location of the primer, the negative strand (−) of DNA can be used for primer design if positive strand (+) is difficult to assay; (2) Design primers of 18 nucleotides in length or greater with melting temperatures of 45° C. or greater; (3) Check primers for extendable hairpin structures and primer dimer formation; (4) Primers should be PAGE purified; (5) A negative control (lacking DNA template) should be run when evaluating a new primer.
We also tested whether JDF-3 P410L/A485 could be used for multiplexing (several primers with one template in a single reaction) without the signal strength being affected.
We then determined the amount of SAP or CIAP that can be used to purify minisequencing products from unincorporated rhodamine labeled-ddNTPs. 0.5 unit of SAP or I unit of CIAP in 10 μl minisequencing reactions degrades all unincorporated dye-ddNTPs. Using suboptimal units of alkaline phosphatase could result in fluorescent signal from unincorporated dye-ddNTPs, thereby masking the SNP signal.
In optimizing the kit cycling conditions, we evaluated whether thermal cycling improved product yield. As
Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.
This application is a continuation-in-part of application U.S. Ser. No. 09/698,341, pending.
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| 5674679 | Fuller | Oct 1997 | A |
| 5882904 | Riedl et al. | Mar 1999 | A |
| 6027913 | Sommer | Feb 2000 | A |
| 20040081965 | Sorge et al. | Apr 2004 | A1 |
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| 9739150 | Oct 1997 | WO |
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| Number | Date | Country | |
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| 20030157483 A1 | Aug 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60162600 | Oct 1999 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09698341 | Oct 2000 | US |
| Child | 09896923 | US |
