Patent Grant
11208691
This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “181203_78341-AAAAA-PCT-US_Substitute_Sequence_Listing_CS.txt” which is 5.08 kilobytes in size, and which was created Dec. 3, 2018 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Dec. 3, 2018 as part of this application.
Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The completion of the Human Genome Project (HGP) in early 2000 (1) was a monumental achievement with incredible amount of combined efforts among genome centers and scientists worldwide. The engine behind this decade long project was the Sanger sequencing method, which still currently maintains as the staple of large-scale genome sequencing methodology in high-throughput genome sequencing centers. The main reason behind this prolonged success was in the basic and efficient, yet elegant method that is Sanger dideoxy chain terminating reaction (2). With incremental improvements in this DNA sequencing technology including the use of laser induced fluorescent excitation of energy transfer dyes (3), engineered DNA polymerases (4) and capillary electrophoresis (5) as well as in the areas of sample preparation, informatics, and sequence analysis software (6-9), the Sanger sequencing platform has been able to maintain its status as champion in the sequencing world. Current state-of-the-art Sanger based DNA sequencers can produce over 700 bases of clearly readable sequence in a single run from templates up to 30 kb in length (10-12). However, as is with most of technological inventions, the continual improvements in this sequencing platform has come to a stagnant plateau, with the current cost estimate for producing a high-quality microbial genome draft sequence at around $10,000 per megabase pair. Current DNA sequencers based on the Sanger method allow up to 384 samples to be analyzed in parallel.
While fluorescent-based SBS methods have almost unlimited ability for parallelization, restricted only by the resolution of the imaging system, to date they have been limited to read lengths of about 35 bases. The successful implementation of sequencing by synthesis (SBS) is effectively dependent on the read length of the target DNA template. One of the major factors that determines the read length when performing SBS is the number of available templates. Our laboratory has recently developed two powerful approaches for SBS: 1) Hybrid SBS with nucleotide reversible terminator (NRTs, 3′-O—R1-dNPTs) in combination with fluorescently labeled dideoxynucleotide (ddNTPs-R2-fluorophore), and 2) SBS with cleavable fluorescent nucleotide reversible terminator (C-F-NRTs, 3′-O—R1-dNTPs-R2-fluorophore). (“Four-color DNA Sequencing with 3′-O-modified Nucleotide Reversible Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides”. J. Guo, N. Xu, Z. Li, S. Zhang, J. Wu, D. Kim, M. S. Marma, Q. Meng, H. Cao, X. Li, S. Shi, L. Yu, S. Kalachikov, J. Russo, N.J. Turro, J. Ju. Proceedings of the National Academy of Sciences USA. 2008, 105, 9145-9150) (“Four-Color DNA Sequencing by Synthesis Using Cleavable Fluorescent Nucleotide Reversible Terminators”. J. Ju, D. Kim, L. Bi, Q. Meng, X. Bai, Z. Li, X. Li, M. S. Marma, S. Shi, J. Wu, J. R. Edwards, A. Romu, N.J. Turro. Proceedings of the National Academy of Sciences USA. 2006, 103, 19635-19640). Since the incorporation of ddNTPs-R2-fluorophore into a strand of DNA permanently terminates further extensions of that template in the first approach and the incorporation and cleavage of C-F-NRTs leaves a tail of the modified nucleotide that causes possible steric hindrance to lower the incorporation efficiency of the subsequent base in the second approach, the total number of sequenceble templates decreases after each cycle of SBS reaction. Various means can be employed to minimize this rate of template reduction. Among those, a powerful method termed template “walking” can potentially diminish the negative effect of template termination or reduction and extend the read length of SBS at least two to three-fold.
A composition is provided having a first, second and third portion wherein the second portion has the following structure:
wherein α represents a point of attachment to the first portion and β represents a point of attachment to the third portion.
A method is provided for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
A method is provided for determining the identity of each of a series of consecutive nucleotide residues in a self-priming nucleic acid comprising:
A kit is provided for use in sequencing a nucleic acid comprising:
An array is provided comprising a nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
An array is provided comprising a self-priming nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
A method is provided for increasing a read length of DNA sequencing by synthesis comprising (a) providing deoxynucleotide triphosphate analogues wherein the deoxynucleotide triphosphate analogues differ from deoxynucleotide triphosphates by having a methylazido group attached to a 3′ O atom thereof and by having a detectable marker attached to a 1 nitrogen or a 9 nitrogen of a base thereof through a linker comprising the structure
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
A method for determining the identity of each of a series of consecutive nucleotide residues in a self-priming nucleic acid comprising:
A method for determining the identity of each of a series of consecutive nucleotide residues in a plurality of nucleic acids comprising, the same series of consecutive nucleotides comprising:
A method for determining the identity of consecutive nucleotide residues in a self-priming nucleic acid comprising:
A kit for use in sequencing a nucleic acid comprising:
An array comprising a nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
An array comprising a self-priming nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
A method is provided for increasing a read length of DNA sequencing by synthesis coupled with Sanger dideoxynucleotide terminating reaction (a) providing deoxynucleotide triphosphate analogues wherein the deoxynucleotide triphosphate analogues differ from deoxynucleotide triphosphates by having a methylazido group attached to a 3′ O atom thereof and providing dideoxynucleotide triphosphate analogues wherein the dideoxynucleotide triphosphate analogues differ from dideoxynucleotide triphosphates by having a detectable marker attached to a 1 nitrogen or a 9 nitrogen of a base thereof through a linker comprising the structure
As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.
A—Adenine;
C—Cytosine;
DNA—Deoxyribonucleic acid;
G—Guanine;
RNA—Ribonucleic acid;
T—Thymine; and
U—Uracil.
“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).
“Type” of nucleotide refers to A, G, C, T or U. “Type” of base refers to adenine, guanine, cytosine, uracil or thymine.
“Mass tag” shall mean a molecular entity of a predetermined size which is capable of being attached by a cleavable bond to another entity.
“Solid substrate” shall mean any suitable medium present in the solid phase to which a nucleic acid or an agent may be affixed. Non-limiting examples include chips, beads and columns.
“Hybridize” shall mean the annealing of one single-stranded nucleic acid to another nucleic acid based on sequence complementarity. The propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.)
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range.
Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
A composition is provided having a first, second and third portion wherein the second portion has the following structure:
wherein α represents a point of attachment to the first portion and β represents a point of attachment to the third portion.
α may be directly linked to the first portion, e.g. comprising a base, or bonded to the, for example base via, e.g. an alkynylene. β may be directly linked to the third portion, e.g. a detectable marker, or bonded to the third portion, for example via another group.
In an embodiment the first portion is a deoxynucleotide or a dideoxynucleotide and the third portion is a detectable marker. In an embodiment the detectable marker is a fluorescent dye. In an embodiment the deoxynucleotide or dideoxynucleotide comprises a methylazido group attached to a 3′ O atom thereof.
In an embodiment the composition has the structure:
wherein the dye in each structure is a fluorescent dye.
In an embodiment the composition has the structure:
A method is provided for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
A method is provided for determining the identity of each of a series of consecutive nucleotide residues in a self-priming nucleic acid comprising:
In an embodiment of the instant methods, steps b) and c) can be performed simultaneously, or in the order step b) then step c) or in the order step c) then step b). In an embodiment of the instant methods, the nucleic acid is DNA and the nucleic acid polymerase is a 9° N thermopolymerase. In an embodiment of the instant methods, the cleavable chemical group is a methylazido group. In an embodiment of the instant methods, the four dNTP analogues have the following structures:
In an embodiment the four dNTP analogues have the following structures:
In an embodiment up to 1000 consecutive nucleotides are identified. In an embodiment up to 1×104 consecutive nucleotides are identified. In an embodiment up to 1×106 consecutive nucleotides are identified. In an embodiment the nucleic acid is immobilized on a solid surface. In an embodiment the solid surface is a chip or a bead.
A kit is provided for use in sequencing a nucleic acid comprising:
In an embodiment four nucleotide analogues having the following structures:
An array is provided comprising a nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
An array is provided comprising a self-priming nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ 0 atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
In embodiment the detectable marker is a fluorophore.
A method is provided for increasing a read length of DNA sequencing by synthesis comprising (a) providing deoxynucleotide triphosphate analogues wherein the deoxynucleotide triphosphate analogues differ from deoxynucleotide triphosphates by having a methylazido group attached to a 3′ O atom thereof and by having a detectable marker attached to a 1 nitrogen or a 9 nitrogen of a base thereof through a linker comprising the structure
wherein α represents a point of attachment to a the base and β represents a point of attachment to the detectable marker, (b) incorporating a plurality of the deoxynucleotide triphosphate analogues into a nucleic acid being synthesized in the DNA sequencing by synthesis, and (c) cleaving the methylazido and detectable marker from each dNTP analogue, so as to thereby increase the read length of the DNA sequence by synthesis.
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
A method for determining the identity of each of a series of consecutive nucleotide residues in a self-priming nucleic acid comprising:
c) contacting the dNTP analogue which has formed the phosphodiester bond with tris(2-carboxyethyl)phosphine so as to thereby (1) cleave the fluorophore and (2) cleave the cleavable chemical group from the dNTP;
In an embodiment of the instant methods, steps b) and c) can be performed simultaneously, or in the order step b) then step c) or in the order step c) then step b). In an embodiment of the instant methods, the nucleic acid is DNA and the nucleic acid polymerase is a 9° N thermopolymerase. In an embodiment of the instant methods, the cleavable chemical group is a methylazido group. In an embodiment of the instant methods, the four dNTP analogues have the following structures:
In an embodiment the four dNTP analogues have the following structures:
In an embodiment up to 1000 consecutive nucleotides are identified. In an embodiment up to 1×104 consecutive nucleotides are identified. In an embodiment up to 1×106 consecutive nucleotides are identified. In an embodiment the nucleic acid is immobilized on a solid surface. In an embodiment the solid surface is a chip or a bead.
A method for determining the identity of each of a series of consecutive nucleotide residues in a plurality of nucleic acids comprising, the same series of consecutive nucleotides comprising:
A method for determining the identity of consecutive nucleotide residues in a self-priming nucleic acid comprising:
In an embodiment of the instant methods, steps b) and c) can be performed simultaneously, or in the order step b) then step c) or in the order step c) then step b). In an embodiment of the instant methods, the nucleic acid is DNA and the nucleic acid polymerase is a 9° N thermopolymerase. In an embodiment of the instant methods, the cleavable chemical group is a methylazido group.
In an embodiment of the instant methods, the four ddNTP analogues have the following structures:
In an embodiment, the four dNTPs have the following structures:
In an embodiment the four ddNTP analogues have the following structures:
In an embodiment up to 1000 consecutive nucleotides are identified. In an embodiment up to 1×104 consecutive nucleotides are identified. In an embodiment up to 1×106 consecutive nucleotides are identified. In an embodiment the nucleic acid is immobilized on a solid surface. In an embodiment the solid surface is a chip or a bead.
A kit is provided for use in sequencing a nucleic acid comprising:
with
In an embodiment, four dideoxynucleotide analogues having the following structures:
An array is provided comprising a nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ O atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
An array is provided comprising a self-priming nucleic acid attached to a solid surface, wherein the nucleic acid comprises an azidomethyl group attached to a 3′ 0 atom thereof and a molecule having the structure:
wherein α represents a point of attachment to a 3′ base of the nucleic acid and β represents a point of attachment to a detectable marker.
In embodiment the detectable marker is a fluorophore.
A method is provided for increasing a read length of DNA sequencing by synthesis coupled with Sanger dideoxynucleotide terminating reaction (a) providing deoxynucleotide triphosphate analogues wherein the deoxynucleotide triphosphate analogues differ from deoxynucleotide triphosphates by having a methylazido group attached to a 3′ O atom thereof and providing dideoxynucleotide triphosphate analogues wherein the dideoxynucleotide triphosphate analogues differ from dideoxynucleotide triphosphates by having a detectable marker attached to a 1 nitrogen or a 9 nitrogen of a base thereof through a linker comprising the structure
This invention provides the instant method, wherein the detectable bound to the base via a cleavable linker is a dye, a fluorophore, a chromophore, a combinatorial fluorescence energy transfer tag, a mass tag, or an electrophore. Combinatorial fluorescence energy tags and methods for production thereof are disclosed in U.S. Pat. No. 6,627,748, which is hereby incorporated by reference.
Detectable tags and methods of affixing nucleic acids to surfaces which can be used in embodiments of the methods described herein are disclosed in U.S. Pat. Nos. 6,664,079 and 7,074,597 which are hereby incorporated by reference.
This invention also provides the instant method, wherein the primer is a self-priming moiety.
This invention also provides the instant method, wherein the DNA is bound to a solid substrate. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule. This invention also provides the instant method, wherein the DNA is alkyne-labeled. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized. This invention also provides the instant method, wherein the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction. Immobilization of nucleic acids is described in Immobilization of DNA on Chips II, edited by Christine Wittmann (2005), Springer Verlag, Berlin, which is hereby incorporated by reference. This invention also provides the instant methods, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized or the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction. In an embodiment, the DNA or nucleic acid is attached/bound to the solid surface by covalent site-specific coupling chemistry compatible with DNA.
This invention also provides the instant method, wherein the solid substrate is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, or a column. This invention also provides the instant method, wherein the solid substrate is gold, quartz, silica, plastic, glass, nylon, diamond, silver, metal, or polypropylene.
This invention also provides the instant method, wherein the solid substrate is porous. Chips or beads may be made from materials common for DNA microarrays, for example glass or nylon. Beads/micro-beads may be in turn immobilized to chips.
This invention also provides the instant method, wherein about 1000 or fewer copies of the DNA are bound to the solid substrate. This invention also provides the instant invention wherein 2×107, 1×107, 1×106 or 1×104 or fewer copies of the DNA are bound to the solid substrate.
This invention also provides the instant method, wherein the nucleotide analogues comprise one of the fluorophores Cy5, Bodipy-FL-510, ROX and R6G.
This invention also provides the instant method, wherein the DNA polymerase is a 9° N polymerase or a variant thereof. DNA polymerases which can be used in the instant invention include, for example E. coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase™, Taq DNA polymerase and 9° N polymerase (exo-) A485L/Y409V. RNA polymerases which can be used in the instant invention include, for example, Bacteriophage SP6, T7 and T3 RNA polymerases.
Methods for production of cleavably capped and/or cleavably linked nucleotide analogues are disclosed in U.S. Pat. No. 6,664,079, which is hereby incorporated by reference.
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
In an embodiment the linker in each of step a) and j) independently each comprise the structure:
or the structure:
In an embodiment a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.
In an embodiment one or more linkers are photocleavable or chemically cleavable.
In an embodiment one or more chemical groups are photocleavable or chemically cleavable.
In an embodiment R in the structures set forth in steps a) and or j) is independently chosen from a —N3 group or an allyl group.
In an embodiment the cleavable chemical group in step g) is independently chosen from a —N3 group or an allyl group.
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
In an embodiment in steps g) and h) the three types of dNTPs are chosen from the group dATP, dCTP, dGTP, dTTP or dITP.
In an embodiment the linker in each of step a) and j) independently each comprise the structure:
or the structure:
In an embodiment a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.
In an embodiment one or more linkers are photocleavable or chemically cleavable.
In an embodiment one or more chemical groups are photocleavable or chemically cleavable.
In an embodiment R in the structures set forth in steps a) and or j) is independently chosen from a —N3 group or an allyl group.
In an embodiment the cleavable chemical group in step g) is independently chosen from a —N3 group or an allyl group.
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
In an embodiment in step g) the three types of dNTPs are chosen from the group dATP, dCTP, dGTP and dTTP.
In an embodiment the linker in each of step a) and j) independently each comprise the structure:
or the structure:
In an embodiment a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.
In an embodiment one or more linkers are photocleavable or chemically cleavable.
In an embodiment one or more chemical groups are photocleavable or chemically cleavable.
In an embodiment R in the structures set forth in steps a) and or j) is independently chosen from a —N3 group or an allyl group.
In an embodiment the cleavable chemical group in step g) is independently chosen from the a —N3 group or an allyl group.
The methods described herein can be applied mutatis mutandis to sequencing RNA using the appropriate ddNTPS or analogues thereof and dNTPS and analogues thereof.
In the methods, base-pairing complementarity allows the sequence of the extended primer or of the target nucleic to be readily determined.
Dehybridize is understood by those skilled in the art to mean to disassociate the hybridized primer (or extended strand thereof) from the target nucleic acid without destroying the target nucleic acid and thus permitting further hybridization of a second primer to the target nucleic acid. Hybridization as used herein in one embodiment means stringent hybridization, for examples as described in Sambrook, J., Russell, D. W., (2000) Molecular Cloning: A Laboratory Manual: Third Edition. Cold Spring Harbor Laboratory Press
“Type” of dNTP or ddNTP is used to distinguish dNTP or ddNTPs comprising different bases.
All combinations of the various elements described herein are within the scope of the invention.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Azido Modified Nucleotide Analogs
While both 3′-O-Allyl-dNTPs and 3′-O-photocleaveble linker (PC)-dNTPs have offered concrete evidence for their implementation in sequencing by synthesis (SBS), a new set of nucleotide analogs, modified with the small azido group (N3), is investigated to seek potential improvement over the current system. There are several advantages for using azido moiety as 3′ capping group and also as a dye linker (3′-O-Azido-dNTPs-Azido-Dye), first and foremost being the application of extremely mild cleavage conditions. As disclosed herein, an example of the Staudinger reaction, an azido group can be effectively converted into an amine with phosphine in DNA-friendly aqueous solution (35). This efficient reduction is further enhanced through the utilization of Tris(2-Carboxyethyl) phosphine (TCEP), an odorless and stable agent often used to digest peptide disulfide bonds (
Similar to allyl and nitrobenzyl alterations previously reported, two positions of the nucleotide need to be modified with the azido moiety to afford a set of 3′-O-Azido-dNTPs-Azido-Dye. The small azido methyl group (—CH2—N3) is used to cap the 3′ position of the sugar base while a novel azido linker connects unique fluorophores to the 5′ position of C/U and the 7′ position of A/G (see novel structures in
With such a formulation the same reagent (TCEP) can be used to cleave the azido groups at both positions simultaneous, although the mechanisms of cleavage differ slightly. According to Staudinger, TCEP reduces the azido-methyl capping group to methylamine at the 3′ sugar base. Since the carbon of the methylamine is highly unstable due to its position between two electron-withdrawing elements (oxygen and nitrogen), the methylamine is hydrolyzed in the presence of water that recovers the hydroxyl group at the 3′ position. For the azido linker, the same Straudinger reduction takes place. However immediately after the attachment of TCEP to azido, the intermediate attacks the ester bond to afford total cleavage of the fluorophore (
In addition to the dual azido/azido modification, an alternative approach is to attach the fluorophore via a PC (nitrobenzyl) linker while conserving the 3′ capping with the azido methyl group (3′-O-Azido-dNTPs-PC-Dye,
The extension and detection steps for this set of nucleotides are analogous to those for 3′-O-Azido-dNTPs-Azido-Dye. An additional photolysis procedure is involved during the deprotection step. This dual cleavage process might offer different advantages for removing the fluorophore than the Staudinger reduction.
An alternative sequencing method that is a hybrid between the Sanger dideoxy chain terminating reaction, and SBS.
In this approach, four nucleotides, modified as reversible terminators by capping the 3′-OH with a small reversible moiety so that they are still recognized as substrates by DNA polymerase, are used in combination with a small percentage of four cleavable fluorescent dideoxynucleotides to perform SBS. DNA sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. Upon removing the 3′-OH capping group from the DNA products generated by incorporating the 3′-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination (
Using an azidomethyl group as a chemically reversible capping moiety in the 3′-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to ddNTPs, four 3′-O—N3-dNTPs and four ddNTP-N3-fluorophores were synthesized for the hybrid SBS. The azidomethyl capping moiety on the 3′-OH group and the cleavable fluorophore on the DNA extension products are efficiently removed after fluorescence detection for sequence determination using a chemical method that is compatible to DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with read length of over 30 bases using this hybrid SBS method.
Sequence by Synthesis with Template “Walking”
The fundamental rationale behind primer resetting is to regenerate the original primer site or to insert two or more primer sites of known sequences into the target DNA so SBS can be carried out at each site sequentially. In general, three steps are involved with this approach: 1) annealing of the first primer, 2) performing SBS, 3) denaturing the sequenced section of the template to recover a single-stranded DNA for the second primer annealing. These steps are carried out repeatedly until the target DNA is sequenced in its entirety. The advantage of primer resetting lies in its ability to restore all the templates after the denaturation step, including those that are terminated with ddNTPs, so the next cycle of SBS can restart with potentially the same amount of sequenceable DNA as the previous round.
Three approaches for achieving longer read lengths that rely on this template “walking” concept are described. In the first strategy, the DNA sequence is reset by reattaching the original primer, extending the chain with natural or minimally modified nucleotides to the end of the first round sequence, and then sequencing from that point. The second strategy relies on annealing of a second round primer that is longer than the first, containing at its 5′ end the same sequence as the original primer, followed by a run of 20 universal nucleotides such as inosine, from which the second round of sequencing can be primed. If the duplex stability of this highly degenerate primer with DNA templates is found to be low, a number of locked nucleotides can be added at either end of the primer to increase the stability of the primer-template complex. In the third strategy, extra priming sites are inserted within a template strand via Type IIS or Type III restriction-recircularization. Each of these approaches has distinct advantages and some difficulties that need to be overcome. None of the three aforementioned strategies are sensitive to the type of library (genomic, cDNA or other), to the method of amplification prior to sequencing (spotting of clones, ePCR, polony PCR), or the mode of sequencing (Hybrid SBS and SBS with C-F-NRTs). Hence they are all sequence unbiased, thus greatly increasing their range of applications in sequencing technologies.
Results
Solution Extension with 3′-O-Azido-dNTPs
To verify the feasibility of using azido-modified nucleotides in SBS, a set of 3′-O-Azido-dNTPs (
Each nucleotide analog (3′-O-Azido-dATP, mw=541; 3′-O-Azido-dGTP, mw=558; 3′-O-Azido-dCTP, mw=518; 3′-O-Azido-dTTP, mw=533) was incorporated into its corresponding looped primer in solution with manganese (Mn2+, 20 mM) and the mutant 9° N Thermopolymerase. The extensions were carried out at 65° C. for various time spans ranging from 20 minutes to 5 minutes. Even with the shortest reaction time of 5 minutes, 100% incorporation was confirmed with MALDI-TOF mass spectroscopy (MS) by observing the total disappearance of primer peak (m/z=7966) and the emergence of extended product peak (˜m/z=8320,
3′-O-Azido-dNTPs-Azido Linker-Dye
Solution Extension with 3′-O-Azido-dNTPs-Azido-Dye
Immediately after establishing the protocol to work with azido-modified nucleotides in SBS, synthetic work for the set of 3′-O-Azido-dNTPs-Azido Linker-Dye was set forth. Two nucleotides, 3′-O-Azido-dCTP-PC-BodipyFL and 3′-O-Azido-dUTP-PC-R6G were successfully synthesized and characterized. To test the incorporation of each nucleotide, extension reactions in solution, similar to those with 3′-O-Azido-dNTPs, were carried out with looped primer (m/z=7966), 9° N Thermopolymerase enzyme, and Mn2+ at 65° C. for 25 minutes. The products were verified through MALDI-TOF MS (
Surface Extension with 3′-O-Azido-dNTPs-Azido Linker-Dye
As a part of the preliminary study, the two chemically cleavable fluorescent nucleotide analogs were used in an SBS reaction to identify the sequence of a self-primed DNA template (130 base pairs) immobilized on a solid surface. A reaction mixture of 3′-O-Azido-dCTP-Azido-BodipyFL, 3′-O-Azido-dUTP-Azido-R6G, 3′-O-Azido-dATP, and 3′-O-Azido-dGTP were prepared for the incorporation. A synchronization step was performed with the full set of 3′-O-Azido-dNTPs after incorporation to extend any remaining priming strand. After detection of the fluorescent signal, the chip surface was immersed in reduction solution (100 mM TCEP, pH=10) and incubated for 25 minutes at 65° C. to cleave both the fluorophore and 3′-O-Azido group. Upon confirmation of the removal of fluorophore, the cycles of extension, detection, and reduction were repeated to sequence the following bases (
SBS on Surface with 3′-O-Azido-dNTPs-PC/Azido-Dye
Upon completing the synthesis of both 3′-O-Azido-dNTPs-PC-Dye and 3′-O-Azido-dNTPs-Azido-Dye sets, sequencing by synthesis of DNA templates attached on solid surface will be carried out. By carefully optimizing incorporation and cleavage conditions, the goal will be the achievement of maximum base read length of each template with high consistency.
PCR Product Attachment on Beads
To expand the efficiency and increase the throughput of SBS, it is ideal to attach large quantities of different DNA templates to solid surface so that each one of these templates can be sequenced during a single cycle of SBS simultaneously. One approach will be to affix PCR product on a single bead, and then immobilize copious amount of such beads on one glass chip. Various types of beads, such as magnetic, melamine, and sepharose, will be tested in order to select one with efficient attachment to surface, durable stability during reaction cycles, and minimal unspecific absorption of fluorophores. By coupling the micro-beads chip with the azido modified reversible terminator nucleotides, SBS technology will reach the next plateau of high-throughput DNA sequencing.
Design and Synthesis of 3′-O-Modified NRTs and Cleavable Fluorescent Dideoxynucleotide Terminators for the Hybrid SBS.
Four 3′-O-azidomethyl-modified NRTs (3′-O—N3-dNTPs) (
To demonstrate the feasibility of carrying out the hybrid SBS on a DNA chip, four cleavable fluorescent dideoxynucleotides ddNTP-N3-fluorophores (ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) were synthesized (
Continuous Polymerase Extension by Using 3′-O-Modified NRTs and Characterization by MALDI-TOF Mass Spectrometry
To verify that the 3′-O—N3-dNTPs incorporate accurately in a base specific manner, four continuous DNA extension reaction and cleavage were carried out in solution by using 3′-O—N3-dNTPs as substrates. This allowed the isolation of the DNA product at each step for detailed molecular structure characterization as shown in
Polymerase Extension by Cleavable Fluorescent Dideoxynucleotide Terminators and Characterization by MALDI-TOF Mass Spectrometry
To verify that the four cleavable fluorescent ddNTPs (ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) (
4-Color DNA Sequencing by Synthesis on a Chip Using Photocleavable Fluorescent Dideoxynucleotide/3′-Modified Photocleavable Nucleotide Combination Remnant of Sanger Sequencing.
In the four-color hybrid SBS approach, the identity of the incorporated nucleotide is determined by the unique fluorescent emission from the four fluorescent dideoxynucleotide terminators, while the role of the 3′-O-modified NRTs is to further extend the DNA strand to continue the determination of the DNA sequence. Therefore, the ratio between the amount of ddNTP-N3-fluorophores and 3′-O—N3-dNPTs during the polymerase reaction determines how much of the ddNTP-N3-fluorophores incorporate and thus the corresponding fluorescent emission strength. With a finite amount of immobilized DNA template on a solid surface, initially the majority of the priming strands should be extended with 3′-O—N3-dNPTs, while a relative smaller amount should be extended with ddNTP-N3-fluorophores to produce fluorescent signals that are above the fluorescent detection system's sensitivity threshold for sequence determination. As the sequencing cycle continues, the amount of the ddNTP-N3-fluorophores need to be gradually increased to maintain the fluorescence emission strength for detection. Following these guidelines, we performed the hybrid SBS on a chip-immobilized DNA template using the 3′-O—N3-dNTP/ddNTP-N3-fluorophore combination and the results are shown in
De novo sequencing reaction on the chip was initiated by extending the self-priming DNA by using a solution consisting of four 3′-O—N3-dNPTs and four ddNTP-N3-fluorophores, and 9° N DNA polymerase. The hybrid SBS allows for the addition of all eight, nucleotide substrates simultaneously to unambiguously determine DNA sequences. This reduces the number of steps needed to complete the sequencing cycle, while increasing the sequencing accuracy because of competition among the substrates in the polymerase reaction. The DNA products extended by ddNTP-N3-fluorophores, after fluorescence detection for sequence determination and cleavage, are no longer involved in the subsequent polymerase reaction cycles because they are permanently terminated. Therefore, further polymerase reaction only occurs on a DNA strand that incorporates the 3′-O—N3-dNPTs, which subsequently turn back into natural nucleotide on cleavage of the 3′-OH capping group, and should have no deleterious effect on the polymerase binding to incorporate subsequent nucleotides for growing the DNA chains. However, successive addition of the previously designed cleavable fluorescent NRTs (22, 37, 38) into a growing DNA strand during SBS leads to a newly synthesized DNA chain with a leftover propargyl amino group at each nucleobase. This may interfere with the ability of the enzyme to efficiently incorporate the next incoming nucleotide, which will lead to loss of synchrony and thereby reduction in the read length. This challenge might potentially be overcome by reengineering DNA polymerases that efficiently recognize and accept the modified DNA strand, or by alternative design of the fluorescent NRTs (39).
To negate any lagging fluorescence signal that is caused by a previously unextended priming strand, a synchronization step was added to reduce the amount of unextended priming strands after the initial extension reaction shown in the scheme of
The four-color images from a fluorescence scanner for each step of the hybrid SBS on a chip is shown in
Strategy 1: Template “Walking” by Unlabeled Nucleotides
The fundamental rationale behind this template “walking” strategy is the removal of the sequenced strand and reattaching of the original primer to allow the extension, or walking, of the template with a combination of natural and modified nucleotides to the end of the first round sequence so that SBS can be carried out from that point. Since the original sequenced strand is stripped away, including those terminated with ddNTPs, all the templates become available for “walking”. Given that “walking” is carried out with either natural or 3′-modified nucleotides, the subsequent round of SBS is performed on nascent DNA strands for maximum read length. The advantage of template “walking” is its ability to restore all the templates after the denature step, includes those that are terminated with ddNTPs, so the next cycle of SBS can restart with potentially the same amount of nascent DNA as the previous round. The “walking” methodology is applicable to both hybrid SBS and SBS with C-F-NRTs, and has the potential to dramatically increase the read lengths of these SBS technologies (
Template “Walking” for Hybrid SBS
1. Hybrid SBS (1st Round)
DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to efficiently decipher multiple DNA sequences in parallel. Hybrid SBS is a hybrid DNA sequencing method between the Sanger dideoxy chain terminating reaction and SBS. In this approach, four nucleotides (
2. Template “Walking”
Immediately after the first round of SBS, all of the elongated primers ended terminated with ddNTPs are removed from the template by denaturing. The templates are freed again and available for further sequencing reactions. To achieve template “walking”, the same starting primer is annealed to the template again and enzymatic incorporation is conducted to fill the gap between first and second stages of SBS. Five strategies are available for the walking process. Each approach has its advantages and shortcomings, which are summarized in the following.
Method 1. Nucleotide reversible terminators (3′-O—R1-dNTPs) are used as substrates to perform enzymatic incorporation (
3. Re-Initiation of Hybrid SBS
Once the “walking” process is completed, the second stage of SBS is conducted using mixture of nucleotide reversible terminators and fluorescently labeled dideoxynucleotides as incorporation substrates same as described above. Another cluster of bases on the template can be continuously revealed, leading to the doubling of the original read length. The SBS-walking-SBS process is repeated to generate maximum read length.
1. Design and Synthesis of 3′-O-Modified NRTs and Cleavable Fluorescent Dideoxynucleotide Terminators for the Hybrid SBS
Four 3′-O-azidomethyl-modified NRTs (3′-O—N3-dNPTs) were synthesized and evaluated (
To demonstrate the feasibility of carrying out the hybrid SBS on a DNA chip, four cleavable fluorescent dideoxynucleotide terminators were designed and synthesized, ddNTP-N3-Fluorophores (ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX and ddGTP-N3-Cy5) (
2. Four-Color DNA Sequencing on a Chip by the Hybrid SBS Approach
Hybrid SBS was performed on a chip-immobilized DNA template using the 3′-O—N3-dNTP/ddNTP-N3-fluorophore combination and the results are shown in
The four-color images from a fluorescence scanner for each step of the hybrid SBS on a chip is shown in
3. Primer Reset and 2nd Round SBS
To demonstrate the concept of walking, the same self-priming DNA was immobilized on surface as template. After identifying the first 32 bases unambiguously with no errors by the first round hybrid SBS, the primer was reset for the second round SBS by elongating the original primer over the sequenced region via enzymatic incorporations. A solution containing dATP, dTTP, dCTP and 3′-O—N3-dGTP was used to perform the polymerase reaction. 9° N DNA polymerase incorporates 3′ unblocked nucleotides more efficiently, leading to certain percentage of primers not fully extended by 3′-O—N3-dGTP. To minimize this effect, a synchronization step was added to reduce the amount of out-of-phase primers after the initial extension reaction. A synchronization reaction mixture consisting of just 3′-O—N3-dGTP in relative high concentration was used along with the 9° N DNA polymerase. The 3′-O-azidomethyl group on the DNA extension product generated by incorporating 3′-O—N3-dGTP was efficiently removed by using aqueous Tris(2-carboxy-ethyl) phosphine (TCEP) solution to yield a free 3′-OH group for elongating the DNA chain in subsequent cycles of enzymatic incorporation. The entire process of incorporation, synchronization and cleavage were conducted repeatedly until the sequenced bases during the first round SBS were “walked” over. After the primer was reset by the enzymatic incorporation, the second stage of SBS was conducted using mixture of nucleotide reversible terminators and fluorescently labeled dideoxynucleotides as incorporation substrates same as described above. Another 13 bases were successfully identified after template “walking” (
Template “Walking” for SBS with CFNRTs
1. SBS with C-F-NRTs
DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to efficiently decipher multiple DNA sequences in parallel. Disclosed is the development of a DNA sequencing method that involves the extension of target DNA strand with modified cleavable fluorescent nucleotide reversible terminators (C-F-NRTs, 3′-O—R1-dNTPs-R2-fluorophore) in combination with cleavable nucleotide reversible terminators (C-NRTs, 3′-O—R1-dNTPs). A set of four C-F-NRTs is produced via dual modifications by capping the 3′-OH group with a small chemical moiety and tethering a fluorophore through a cleavable linker to either the 7-position of the purines (A, G) or the 5-position of the pyrimidines (C, T) so that they are still recognized as substrates by DNA polymerase. Another set of four C-NRTs is modified similarly as the C-F-NRTs except no fluorophore is attached, which results in a reduction of the size of C-NRTs and the increment of DNA polymerase incorporation efficiency. In this approach, an extension mixture composed of the C-NRTs with a small percentage of the C-F-NRTs is used to perform SBS. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by the C-F-NRTs. Immediately following the detection step, a synchronization reaction is performed using only the C-NRTs to extend the un-extended DNA strands. A dideoxynucleotides (ddNTPs) capping step is carried out afterwards to completely rid of the remaining un-extended DNA. Upon removing the 3′-OH capping group from the DNA products generated by incorporating both C-F-NRTs and C-NRTs and the fluorophore from the C-F-NRTs, the polymerase reaction reinitiates to continue the sequence determination. The following scheme (
Four 3′-O—N3-dNTPs-N3-fluorophore (
After fluorescence detection for sequence determination, the azidomethyl capping moiety on the 3′-OH and the fluorophore attached to the DNA extension product via the azido-based cleavable linker are efficiently removed using tris(2-carboxyethyl)phosphine (TCEP) in aqueous solution compatible with DNA. Various DNA templates, including those with homopolymer regions were accurately sequenced with read length of over 20 bases using this SBS method on a chip and a four-color fluorescent scanner (
Four C-F-NRTs (3′-O—N3-dNPTs-N3-fluorophore) were synthesize along with four C-NRTs (3′-O—N3-dNTPs) for the implementation of our four-color de novo DNA sequencing by synthesis approach. During the incorporation stage of SBS, a mixture of the two sets of NRTs is used to extend the DNA strand. Only a small percentage of the 3′-O—N3-dNPTs-N3-fluorophore is used in the mixture so that the majority of the product is extended with the less bulky 3′-O—N3-dNPTs. This approach leads to a more efficient DNA polymerase reaction since the smaller 3′-O—N3-dNTPs are much easier to incorporate. Another advantage of having most of the DNA extended with 3′-O—N3-dNTPs is the fact that after cleavage of the 3′-OH capping group on the product, nascent strand of DNA that have no traces of modification is restored. Such DNA does not have any adverse effect on the DNA polymerase during the subsequent incorporation of the complementary nucleotide. For DNA extended with the 3′-O—N3-dNTPs-N3-fluorophore, which serve as the signal producer, the 3′-OH is also restored after the cleavage step so that the next stage of SBS can be carried out. Therefore, it is possible to recover all the DNA templates after each round of sequencing, dramatically increasing the potential read-length of our SBS methodology. After the incorporation reaction, two separate capping steps, first with 3′-O—N3-dNTPs and then with ddNTPs, are performed. The rationale behind the first capping reaction is to maximize the amount of extension products and to ensure the minimal loss of templates. In case there is any un-extended product after the first capping step, the second capping with ddNTPs is mostly likely to permanently terminate these DNA strands so that all templates are synchronized. Without these precautionary synchronization procedures, mixed fluorescent signals will prevent the identification of the correct nucleotide incorporated. Since both 3′-O—N3-dNTPs-N3-fluorophore and 3′-O—N3-dNTPs are reversible terminators, which allow the sequencing of each base in a serial manner, they can accurately determine the homopolymeric regions of DNA. In addition, due to the fact that all of the steps of our SBS approach are performed on a DNA chip, there is no longer a need for electrophoretic DNA fragment separation as in the classical Sanger sequencing method.
Even though theoretically SBS with C-F-NRTs can be executed without losing templates, the utilization of ddNTPs capping does reduce the number of available templates during the actual sequencing reaction. In addition, the incorporation and cleavage of C-F-NRTs leave a tail on the modified nucleotides that can potentially reduce the incorporation efficiency the subsequent base. Hence template “walking” can be applied to increase read length for this SBS methodology.
2. Template “Walking”
Immediately after the first round of SBS, DNA templates are denatured by heat or mild alkali conditions to rid of the extended primer. The same original primer is re-hybridized to the template chain, and one of the five “walking” methods described in the previous section can be applied to reset the start point for the next round of SBS at the end of the first sequencing run (
3. Re-initiation of SBS with C-F-NRTs
Once the “walking” process is completed, the primer is extended to the end of the previous round of SBS. At this point, hybrid SBS is carried out to identify the subsequent bases. If the process can be repeated more times, it should be theoretically possible to achieve long and significant read length.
Strategy 2: Template “Walking” with Universal Bases
In this variation on the Strategy 1, the reset is achieved not with nucleotide walking, but with the use of a longer primer partially consisting of universal nucleotides for the second round. Attachment of the template DNA to the surface and the first few steps of the procedure are identical to the first method. However, after stripping the first extended primer for the initial 20 base readout, a long primer with the following features will be hybridized to the template: (a) the first half is identical to the initial primer; (b) the second half is composed almost entirely of universal bases. One possible candidate for the universal base is inosine, which, in its deoxynucleoside form, can base pair with all four nucleotides, though its affinity for C and A is significantly higher than for G and T; a second possibility is 5-nitroindole; (c) the last one or two anchoring bases of the long primers are degenerate with each of the four possible bases being represented. Because the universal bases can form hydrogen bonds with any of the other four bases with some efficiency, they have the capacity to bind to the first 20 or so bases of the sequence. However, the melting temperature of the ensuing hybridization is reduced substantially by the run of inosines, a few of the bases in the first half and the two 3′-anchoring bases can be substituted with locked nucleotides. Locked nucleic acids have a chemical bond between the 2′ and 4′ carbons of the ribose. While slower to associate with their complementary base, once hybridized, they tend not to dissociate. Thus, they provide a nice solution to ensure that the long primer remains attached appropriately to the template. In addition, the percentage of locked nucleosides in the primer can be manipulated to achieve higher hybridization strength. After hybridization of the above long primer, a second round of either Hybrid SBS or SBS with C-F-NRTs can be performed (
An alternative approach to Strategy 2 is the use of a detachable loop primer, possibly with a labile sugar and glycosylase treatment. After the first round of sequencing, the loop is removed by enzymatic cleavage and denaturation, and then a new identical loop is attached. In a modification that is a composite of “walking” Strategy 1 and 2, the new loop primer can be composed of an initial portion identical to the first loop primer, a “loop out” region that bypasses the first set of sequenced nucleotides, and a degenerate anchoring nucleotide to initiate the second round of sequencing.
Strategy 3: Multiple Primers “Walking”
In this third strategy, one or two additional primer annealing sites are introduced into the DNA to be sequenced at a distance just about equal to the number of bases that can be sequenced from the first primer.
As illustrated in
Several novel modifications of this approach can address the desire of many investigators to sequence an entire 100-base stretch of DNA, the length of a typical exon including surrounding intronic bases adjacent to the splice site. For instance, one can prepare a construct with two internal primers. In this case, the initial vector will be designed with MmeI at one flank and EcoP15I on the other; using two consecutive restriction, cloning and circularization steps, the final construct will consist of four alternative priming sites (two on the insert flanks and two internal), which in the case of 100 bp segments of genomic DNA will guarantee their complete sequencing with 25-30 cycles of SBS and three primer resets. The extra cycles would enable some of the sequence reads to run into the next primer, which would help to confirm the direction (e.g., the last sequence might end with the MmeI or EcoP15I site. Other tricks would include modifying the ends of the primers to allow looping and reverse direction sequencing, incorporation of one or two decoding bases in the internal primers to confirm directions, and deconvoluting the results after all the data is generated. One would want to have a single set of primers for sequencing, regardless of which strand is attached. In order to achieve this, and to overcome the non-directional nature of their insertion, the internal primer or primers will be designed as palindromes so that sequencing can be initiated in either direction.
Materials and Methods
Synthesis of ddCTP-N3-Bodipy-FL-510
Synthesis of ddUTP-N3-R6G
Synthesis of ddATP-N3-ROX
Synthesis of ddGTP-N3-Cy5
Continuous DNA Polymerase Reaction Using Four 3′-O-Modified Cleavable Nucleotides as Reversible Terminators in Solution.
The four NRTs (3′-O—N3-dATP, 3′-O—N3-dCTP, 3′-O—N3-dGTP and 3′-O—N3-dTTP) have been characterized, by performing four continuous DNA-extension reactions sequentially using a self-priming DNA template (5′-ATCGGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:1). The four nucleotides in the template immediately adjacent to the annealing site of the primer are 3′-GCTA-5′, which allows the evaluation of the incorporation and cleavage efficiency of the 4 NRTs. First, a polymerase extension reaction using a pool of all four NRTs along with the self-priming DNA template was performed producing a single base extension product. The reaction mixture for this, and all subsequent extension reactions, consisted of 80 pmol of self-priming DNA template, 160 pmol of 3′-O—N3-dNTPs, lx Thermopol II reaction buffer (New England Biolabs), 40 nmol of MnCl2 and 1 unit of 9° N DNA polymerase (exo-) A485L/Y409V (New England Biolabs) in a total reaction volume of 20 μl. The reaction consisted of incubation at 94° C. for 5 min, 4° C. for 5 min, and 65° C. for 20 min. Subsequently, the extension product was desalted by using a ZipTip and analyzed by Voyager DE MALDI-TOF mass spectrometry (Applied Biosystems). For cleavage, the desalted DNA extension product bearing the 3′-O-azidomethyl group was first resuspended with 5 μl of 50 mM EDTA solution to quench the polymerase activity.
This DNA solution was then mixed with 10 μl of 225 mM TCEP solution (pH 9.0) and incubated at 65° C. for 15 min to yield a cleaved DNA product which was characterized by MALDI-TOF MS. The DNA product with the 3′-O-azidomethyl group removed to generate a free 3′-OH group was purified by using an Oligonucleotide Purification Cartridge (Applied Biosystems) and used as a primer for a second extension reaction using 3′-O—N3-dNTPs. The second extended DNA product was then purified by ZipTip and cleaved as described above. The third and the fourth extensions were carried out in a similar manner by using the previously extended and cleaved product as the primer.
Polymerase Extension Reaction Using Cleavable Fluorescent Dideoxynucleotide Terminators in Solution and Characterization by MALDI-TOF MS.
The four cleavable fluorescent dideoxynucleotide terminators, ddNTP-N3-fluorophores (ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) have been characterized, by performing four separate DNA-extension reactions, each with a different self-priming DNA template allowing the four ddNTP analogues to be incorporated. The resulting DNA extension products were analyzed by MALDI-TOF MS. The following four self-priming DNA templates (26-mer hairpin DNA with a 4-base 5′-overhang) were used for the extension: 5′-GATCGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:2) for ddATP-N3-ROX; 5′-ATCGGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:3) for ddCTP-N3-Bodipy-FL-510; 5′-GATCGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:4) for ddGTP-N3-Cy5; and 5′-GATCGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:5) for ddUTP-N3-R6G. Each of the extension reactions consisted of all four ddNTP-N3-fluorophores (e.g., 120 pmol each of ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) along with 60 pmol of the self-priming DNA template, 1× Thermopol II reaction buffer, 40 nmol of MnCl2 and 1 unit of 9° N DNA polymerase (exo-) A485L/Y4Q9V in a total reaction volume of 20 μl. The reaction consisted of incubations at 94° C. for 5 min, 4° C. for 5 min, and 65° C. for 20 min. Subsequently, the extension product was purified by reverse-phase HPLC using established procedures (40). The fraction containing the desired product was collected and freeze-dried for analysis by MALDI-TOF MS and cleavage. For cleavage of the DNA extension product bearing the ddNTP-N3-fluorophores, the purified DNA product was resuspended in 50 ml of 100 mM TCEP solution (pH 9.0) at 65° C. for 15 min and then analyzed by MALDI-TOF MS.
4-Color DNA Sequencing by Synthesis on a Chip Using Photocleavable Fluorescent Dideoxynucleotide/3′-Modified Photocleavable Nucleotide Combination Remnant of Sanger Sequencing.
Ten microliters of a solution consisting of ddCTP-N3-Bodipy-FL-510 (10 fmol), ddUTP-N3-R6G (20 fmol), ddATP-N3-ROX (40 fmol), ddGTP-N3-Cy5 (20 fmol), 3′-O—N3-dCTP (22 pmol), 3′-O—N3-dTTP (22 pmol), 3′-O—N3-dATP (22 pmol), 3′-O—N3-dGTP (4 pmol), 1 unit of 9° N mutant DNA polymerase(exo-) A485L/Y409V, 20 nmol of MnCl2 and 1× Thermopol II reaction buffer was spotted on the DNA chip. The nucleotide complementary to the DNA template was allowed to incorporate into the primer at 62° C. for 15 min. To synchronize any unextended templates, an extension solution consisting of 38 pmol each of 3′-O—N3-dTTP, 3′-O—N3-dATP, 3′-O—N3-dGTP and 75 pmol of 3′-O—N3-dCTP, 1 unit of 9° N mutant DNA polymerase(exo-) A485L/Y409V, 20 nmol of MnCl2 and 1× Thermopol II reaction buffer was added to the same spot and incubated at 62° C. for 15 min. After washing with SPSC buffer containing 0.1% Tween 20 for 1 min, the chip was rinsed with dH2O, and then scanned with a 4-color fluorescence ScanArray Express scanner (Perkin-Elmer Life Sciences) to detect the fluorescence signal. To perform the cleavage, the DNA chip was placed inside a chamber filled with 100 mM TCEP (pH 9.0) and incubated at 65° C. for 10 min. After washing the surface with dH2O, the chip was scanned again to measure the background fluorescence signal. This process was followed by the next polymerase extension reaction using the 3′-O—N3-dNTP/ddNTP-N3-fluorophore solution with the subsequent synchronization, washing, fluorescence detection, and cleavage processes performed as described above. The 3′-O—N3-dNTP/ddNTP-N3-fluorophore ratio was adjusted to obtain relatively even fluorescence signals. The above reaction cycles were repeated multiple times to obtain de novo DNA sequencing data on a DNA template immobilized on a chip.
Construction of a Chip with Immobilized Self-Priming DNA Template
The 5′-amino-labeled self-priming DNA template 5′-NH2-CAC-TCA-CAT-ATG-TTT-TTT-AGC-TTT-TTT-AAT-TTC-TTA-ATG-ATG-TTG-TTG-CAT-GCG-ACT-TAA-GGC-GCT-TGC-GCC-TTA-AGT-CG-3′ (SEQ ID No:6) was purchased from IDT (Coralville, Iowa). The DNA template was dissolved at 40 μM in 50 mM sodium phosphate buffer, pH 8.5 and spotted using SpotArray 72 arraying robot (Perkin Elmer) onto high density CodeLink microarray slides (GE Healthcare). After spotting, the slides were incubated at ambient temperature (˜24° C.) for 20 hours in a humid chamber containing saturated sodium chloride solution (˜75% humidity) to allow for 5′-tethering of the spotted amino-modified DNA templates to the slide surface functionalized with succinimide ester groups. Upon the incubation the slides were removed from the humid chamber and stored in vacuum desiccator at room temperature until further use. The principal advantage of the hairpin structure introduced into the 3′-portion of the self-priming DNA template is its higher stability and the increased priming efficiency for DNA polymerases as compared to a separate primer/template complex, which is prone to spontaneous dissociation.
3′-O—N3-dNTP/ddNTP-N3-Fluorophore Ratio Used for Four-Color DNA Sequencing on a Chip
To obtain de novo DNA sequencing data on a DNA template immobilized on a chip, the SBS cycle was repeated multiple times using the combination mixture of solution A consisting of 3′-O—N3-dCTP (3 μM), 3′-O—N3-dTTP (3 μM), 3′-O—N3-dATP (3 μM) and 3′-O—N3-dGTP (0.5 μM) and solution B consisting of ddCTP-N3-Bodipy-FL-510 (50 nM), ddUTP-N3-R6G (100 nM), ddATP-N3-ROX (200 nM) and ddGTP-N3-Cy5 (100 nM) in each polymerase extension reaction. The following volumes of solution A and B in each SBS cycle were used to achieve relatively even fluorescence signals.
Discussion
Four 3′-O-modified cleavable reversible terminator nucleotides (3′-O—N3-dNTPs) along with four fluorescent ddNTPs have been synthesized and characterized, and used them to produce 4-color de novo DNA sequencing data on a chip by Sanger/SBS hybrid sequencing approach that has the following advantages. With the 3′-O—N3-dNTPs, after cleavage of the 3′OH capping group of the DNA extension product, there are no traces of modification left on the growing DNA strand. Therefore, there are no adverse effects on the DNA polymerase for incorporation of the next complementary nucleotide. Second, the cleavable fluorescent ddNTPs and 3′-O—N3-dNTPs permanent and reversible terminators, respectively, which allow the interrogation of each base in a serial manner, a key procedure enabling accurate determination of homopolymeric regions of DNA. In addition, because all of the steps of the nucleotide incorporation, fluorescence detection for sequence determination, cleavage of the fluorophore, and the 3′-O-azidomethyl group are performed on a DNA chip, there is no longer a need for electrophoretic DNA fragment separation as in the classical Sanger sequencing method.
In the four-color hybrid SBS approach, the identity of the incorporated nucleotide is determined by the unique fluorescence emission from the four fluorescent dideoxynucleotides, whereas the role of the 3′-O-modified NRTs is to further extend the DNA strand. Therefore, the ratio of the ddNTP-N3-fluorophores and 3′-O—N3-dNTPs during the polymerase reaction determines how much of the ddNTP-N3-fluorophores incorporate and, thus, the corresponding fluorescence emission strength. With a finite amount of immobilized DNA template on a solid surface, initially the majority of the priming strands should be extended with 3′-O—N3-dNTPs, whereas a relatively smaller amount should be extended with ddNTP-N3-fluorophores to produce sufficient fluorescent signals that are above the fluorescence detection system's sensitivity threshold for sequence determination. As the sequencing cycle continues, the amount of the ddNTP-N3-fluorophores needs to be gradually increased to maintain the fluorescence emission strength for detection.
We have experimentally determined the ratio of the 3′-O—N3-dNTPs and ddNTP-N3-fluorophores to yield sequencing read length of 32 bases. The signal strength at base 32 is as strong as that of the first base (
With sequencing read length from 14 to 30 bases in the next generation DNA sequencing systems, massive parallel digital gene expression analogous to a high-throughput SAGE (41) approach has been reported reaching single copy transcript sensitivity (42), and CHIP-Seq (43-45) based on sequencing tags of ˜25 bases has led to many new discoveries in genome function and regulation. It is well established that millions of different PCR template's can be generated on a solid surface through emulsion PCR or clonal amplification (45, 20). Thus, future implementation of the hybrid SBS approach on a high-density bead array platform will provide a high-throughput and accurate DNA sequencing system with wide applications in genome biology and biomedical research.
This application is a continuation of U.S. Ser. No. 15/596,379, filed May 16, 2017, now allowed, which is a continuation of U.S. Ser. No. 14/859,853, filed Sep. 21, 2015, now U.S. Pat. No. 9,670,539, issued Jun. 6, 2017, which is a continuation of U.S. Ser. No. 13/951,269, filed Jul. 25, 2013, now U.S. Pat. No. 9,175,342, issued Nov. 3, 2015, which is a continuation of U.S. Ser. No. 12/734,227, filed Aug. 30, 2010, now abandoned, which is a § 371 national stage of PCT International Application No. PCT/US2008/011891, filed Oct. 17, 2008, claiming the benefit of U.S. Provisional Application No. 60/999,576, filed Oct. 19, 2007, the contents of each of which are hereby incorporated by reference in their entirety into this application.
This invention was made with government support under HG003582 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| 20190085388 A1 | Mar 2019 | US |
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| 60999576 | Oct 2007 | US |
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