Patent Application
20150159152
The sequence listing is filed with the application in electronic format only and is incorporated by reference herein. The sequence listing text file “vBlock Sequence List” was created on Dec. 9, 2014 and is 33 kb in size.
This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing regions of low complexity, repeating elements, difficult to assemble and clone elements, or variable regions containing mixed bases.
Synthetic DNA sequences are a vital tool in molecular biology. They are used in gene therapy, vaccines, DNA libraries, environmental engineering, diagnostics, tissue engineering and research into genetic variants. Long artificially-made nucleic acid sequences are commonly referred to as synthetic genes; however the artificial elements produced do not have to encode for genes, but, for example, can be regulatory or structural elements. Regardless of functional usage, long artificially-assembled nucleic acids can be referred to herein as synthetic genes and the process of manufacturing these species can be referred to as gene synthesis. Gene synthesis provides an advantageous alternative from obtaining genetic elements through traditional means, such as isolation from a genomic DNA library, isolation from a cDNA library, or PCR cloning. Traditional cloning requires availability of a suitable library constructed from isolated natural nucleic acids wherein the abundance of the gene element of interest is at a level that assures a successful isolation and recovery.
Artificial gene synthesis can also provide a DNA sequence that is codon optimized. Given codon redundancy, many different DNA sequences can encode the same amino acid sequence. Codon preferences differ between organisms and a gene sequence that is expressed well in one organism might be expressed poorly or not at all when introduced into a different organism. The efficiency of expression can be adjusted by changing the nucleotide sequence so that the element is well expressed in whatever organism is desired, e.g., it is adjusted for the codon bias of that organism. Widespread changes of this kind are easily made using gene synthesis methods but are not feasible using site-directed mutagenesis or other methods which introduce alterations into naturally isolated nucleic acids.
As another example, a synthetic gene can have restriction sites removed and new sites added. As yet another example, a synthetic gene can have novel regulatory elements or processing signals included which are not present in the native gene. Many other examples of the utility of gene synthesis are well known to those with skill in the art.
Furthermore, a sequence isolated from genomic DNA or cDNA libraries only provides an isolate having that nucleic acid sequence as it exists in nature. It is often desirable to introduce alterations into that sequence. For example a randomized mutant library can be created wherein random bases are inserted into desired positions and then expressed to find desirable properties relative to the wild type sequence. This approach does not allow for specific placement of degenerate bases. In another example, a gene enriched with repeat sequences could be used for genomic mapping or marking.
Although the cost of synthesizing a large library of genes can be substantial, the ability to optimize or change the characteristics of the encoded enzyme or antibody can result in a powerful biological tool or therapeutic. Recombinant antibodies such as Humira® (Abbot Laboratories, Inc.) are widely used as therapeutics, and many others are used as research tools. Those in the art also appreciate that many commercial proteins, such as enzymes, originated from mutant libraries.
Gene synthesis employs synthetic oligonucleotides as the primary building block. Oligonucleotides are made using chemical synthesis, most commonly using betacyanoethyl phosphoramidite methods, which are well-known to those with skill in the art (M. H. Caruthers, Methods in Enzymology 154, 287-313 (1987)). Using a four-step process, phosphoramidite monomers are added in a 3′ to 5′ direction to form an oligonucleotide chain. During each cycle of monomer addition, a small amount of oligonucleotides will fail to couple (n−1 product). Therefore, with each subsequent monomer addition the cumulative population of failures grows. Also, as the oligonucleotide grows longer, the base addition chemistry becomes less efficient, presumably due to steric issues with chain folding. Typically, oligonucleotide synthesis proceeds with a base coupling efficiency of around 99.0 to 99.2%. A 20 base long oligonucleotide requires 19 base coupling steps. Thus assuming a 99% coupling efficiency, a 20 base oligonucleotide should have 0.9919 purity, meaning approximately 82% of the final end product will be full length and 18% will be truncated failure products. A 40 base oligonucleotide should have 0.9939 purity, meaning approximately 68% of the final end product will be full length and 32% will be truncated failure products. A 100 base oligonucleotide should have 0.9999 purity, meaning approximately 37% of the final product will be full length and 63% will be truncated failure products. In contrast, if the efficiency of base coupling is increased to 99.5%, then a 100 base oligonucleotide should have a 0.99599 purity, meaning approximately 61% of the final product will be full length and 39% will be truncated failure products.
Using gene synthesis methods, a series of synthetic oligonucleotides are assembled into a longer synthetic nucleic acid, e.g. a synthetic gene. The use of synthetic oligonucleotide building blocks in gene synthesis methods with a high percentage of failure products present will decrease the quality of the final product, requiring implementation of costly and time-consuming error correction methods. For this reason, relatively short synthetic oligonucleotides in the 40-60 base length range have typically been employed in gene synthesis methods, even though longer oligonucleotides could have significant benefits in assembly. It is well appreciated by those with skill in the art that use of high quality synthetic oligonucleotides, e.g. oligonucleotides with few error or missing bases, will result in high quality assembly of synthetic genes than the use of lower quality synthetic oligonucleotides.
Some common forms of gene assembly are ligation-based assembly, PCR-driven assembly (see Tian et al., Mol. BioSyst., 5, 714-722 (2009)) and thermodynamically balanced inside-out based PCR (TBIO) (see Gao X. et al., Nucleic Acids Res. 31, e143). All three methods combine multiple shorter oligonucleotides into a single longer end-product.
Therefore, to make genes that are typically 500 to many thousands of bases long, a large number of smaller oligonucleotides are synthesized and combined through ligation, overlapping, etc., after synthesis. Typically, gene synthesis methods only function well when combining a limited number of synthetic oligonucleotide building blocks and very large genes must be constructed from smaller subunits using iterative methods. For example, 10-20 of 40-60 base overlapping oligonucleotides are assembled into a single 500 base subunit due to the need for overlapping ends, and twelve or more 500 base overlapping subunits are assembled into a single 5000 base synthetic gene. Each subunit of this process is typically cloned (i.e., ligated into a plasmid vector, transformed into a bacterium, expanded, and purified) and its DNA sequence is verified before proceeding to the next step. If the above gene synthesis process has low fidelity, either due to errors introduced by low quality of the initial oligonucleotide building blocks or during the enzymatic steps of subunit assembly, then increasing numbers of cloned isolates must be sequence verified to find a perfect clone to move forward in the process or an error-containing clone must have the error corrected using site directed mutagenesis.
Traditional methods for assembly have suffered from shortcomings of being unable to clone low complexity sequence motifs such as repeats, homopolymeric nucleotide runs, and high/low GC sequences. In addition, the ability to generate libraries of high sequence variation at defined sequences is even more problematic. Methods for overcoming these limitations have been developed that are based on the synthesis and incorporation of highly pure long single stranded oligonucleotides, such as Ultramers oligonucleotides (Integrated DNA Technologies, Inc.) into double stranded clonal/non-clonal PCR products (see gBlocks® gene block fragments from Integrated DNA Technologies, Inc.). Once fully assembled, the double stranded material can be subjected to error correction methodologies to improve the fidelity of the end product.
The methods of the invention described herein provide high quality oligonucleotide subunits that are ideal for gene synthesis and improved methods to assemble said subunits into longer genetic elements. Furthermore, the genetic elements can be configured to contain regions of high variability by incorporating degenerate bases, These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The methods include the synthesis of long, double stranded nucleic acid sequences containing regions of low complexity, repeating elements, sequences traditionally difficult to assemble and clone, or variable regions containing mixed bases.
In one embodiment, two or more clonal or non-clonal DNA fragments (“gBlocks” or “gene blocks”) are bound or covalently linked together with an overlapping single stranded oligonucleotide (a “bridging oligonucleotide”) optionally containing a variable region, a repeat region or a combination thereof, to form a larger DNA fragment or variable DNA fragment library. The constructed DNA fragments or libraries themselves can be joined with one or more additional DNA fragments, optionally with a bridging oligonucleotide containing further repeat or variable regions, to make longer fragments in either an iterative fashion or in a single reaction.
The bridging oligonucleotide contains overlap regions where the 3′ and the 5′ portions of the bridging oligonucleotide overlap the DNA fragments (gBlocks). Between the bridging oligonucleotide and each gBlock, the overlap can be completely or partially complementary to one strand of the gBlock, the essential element being the ability for the bridging oligonucleotide to hybridize to a strand of the gBlock and allow for strand extension. The resulting product is a larger DNA fragment comprised of a first gBlock, a double-stranded portion encoding the bridge portion of the bridging oligonucleotide, and a second gBlock (
In a further embodiment, a second bridging oligonucleotide containing a fixed base or mixed base bridge sequence and overlap with the second gBlock and a third gBlock, can be added to incorporate more than one fixed or variable region originating from the bridge sequence into the final DNA fragment or library (
The final DNA fragments or library can then be inserted into vectors, such as bacterial DNA plasmids, and clonally amplified through methods well-known in the art.
In a further embodiment, gene blocks are synthesized or combined in such a manner as to provide 3′ and 5′ flanking sequences that enable the synthetic nucleic acid elements to be more easily inserted into a vector using an isothermal assembly method or other homologous recombination methods.
In another embodiment, a single bridging oligonucleotide can combine more than two gBlocks. The bridging oligonucleotide can be long enough to overlap an entire sufficiently complementary strand of a first gBlock, wherein the bridging oligonucleotide is longer than the first gBlock to have 3′ and 5′ ends that can serve to hybridize to a second gBlock 3′ of the first gBlock and hybridize 5′ to a third gBlock, resulting in a new fragment that encodes for at least three gBlocks as well as the bridge sequences.
In another embodiment, the component oligonucleotide(s) that are employed to synthesize the synthetic nucleic acid elements are high-fidelity (i.e., low error) oligonucleotides synthesized on supports comprised of thermoplastic polymer and controlled pore glass (CPG), wherein the amount of CPG per support by percentage is between 1-8% by weight.
Aspects of this invention relate to methods for synthesis of synthetic nucleic acid elements that may comprise genes or gene fragments. More specifically, the methods of the invention include methods of gene assembly through bridging of adjacent clonal or non-clonal double stranded DNA fragments (gBlocks) with a bridging oligonucleotide that optionally contains degenerate, variable or repeat sequences. The bridging oligonucleotide may include degenerate or mismatch bases within the overlapping regions to alter the sequence of adjacent gBlocks.
The term “oligonucleotide,” as used herein, refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms can be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present invention, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
The terms “raw material oligonucleotide” refers to the initial oligonucleotide material that is further processed, synthesized, combined, joined, modified, transformed, purified or otherwise refined to form the basis of another oligonucleotide product. The raw material oligonucleotides are typically, but not necessarily, the oligonucleotides that are directly synthesized using phosphoramidite chemistry. The term “gBlock” is a broader term to refer to double stranded DNA fragments (of clonal or non-clonal origin), sometimes referred to as gene sub-blocks or gene blocks. The synthesis of gBlocks is described in U.S. application Ser. No. 13/742,959 and is referenced herein in its entirety.
The term “base” as used herein includes purines, pyrimidines and non-natural bases and modifications well-known in the art. Purines include adenine, guanine and xanthine and modified purines such as 8-oxo-N6-methyladenine and 7-deazaxanthine. Pyrimidines include thymine, uracil and cytosine and their analogs such as 5-methylcytosine and 4,4-ethanocytosine. Non-natural bases include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, nitroindole, and 2,6-diaminopurine.
The term “base” is sometimes used interchangeably with “monomer”, and in this context it refers to a single nucleic acid or oligomer unit in a nucleic acid chain.
“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
The oligonucleotides used in the inventive methods can be synthesized using any of the methods of enzymatic or chemical synthesis known in the art, although phosphoramidite chemistry is the most common. The oligonucleotides may be synthesized on solid supports such as controlled pore glass (CPG), polystyrene beads, or membranes composed of thermoplastic polymers that may contain CPG. Oligonucleotides can also be synthesized on arrays, on a parallel microscale using microfluidics (Tian et al., Mol. BioSyst., 5, 714-722 (2009)), or known technologies that offer combinations of both (see Jacobsen et al., U.S. Pat. App. No. 2011/0172127).
Synthesis on arrays or through microfluidics offers an advantage over conventional solid support synthesis by reducing costs through lower reagent use. The scale required for gene synthesis is low, so the scale of oligonucleotide product synthesized from arrays or through microfluidics is acceptable. However, the synthesized oligonucleotides are of lesser quality than when using solid support synthesis (See Tian infra.; see also Staehler et al., U.S. Pat. App. No. 2010/0216648). High fidelity oligonucleotides are required in some embodiments of the methods of the present invention, and therefore array or microfluidic oligonucleotide synthesis will not always be compatible.
In one embodiment of the present invention, the oligonucleotides that are used for gene synthesis methods are high-fidelity oligonucleotides (average coupling efficiency is greater than 99.2%, or more preferably 99.5%). High-fidelity oligonucleotides are available commercially up to 200 bases in length (see Ultramer® oligonucleotides from Integrated DNA Technologies, Inc.). Alternatively, the oligonucleotide is synthesized using low-CPG load solid supports that provide synthesis of high-fidelity oligonucleotides while reducing reagent use. Solid support membranes are used wherein the composition of CPG in the membranes is no more than 8% of the membrane by weight. Membranes known in the art are typically 20-50% (see for example, Ngo et al., U.S. Pat. No. 7,691,316). In a further embodiment, the composition of CPG in the membranes is no more than 5% of the membrane. The membranes offer scales as low as subnanomolar scales that are ideal for the amount of oligonucleotides used as the building blocks for gene synthesis. Less reagent amounts are necessary to perform synthesis using these novel membranes. The membranes can provide as low as 100-picomole scale synthesis or less.
Other methods are known in the art to produce high-fidelity oligonucleotides. Enzymatic synthesis or the replication of existing PCR products traditionally has lower error rates than chemical synthesis of oligonucleotides due to convergent consensus within the amplifying population. However, further optimization of the phosphoramidite chemistry can achieve even greater quality oligonucleotides, which improves any gene synthesis method. A great number of advances have been achieved in the traditional four-step phosphoramidite chemistry since it was first described in the 1980's (see for example, Sierzchala, et al. J. Am. Chem. Soc., 125, 13427-13441 (2003) using peroxy anion deprotection; Hayakawa et al., U.S. Pat. No. 6,040,439 for alternative protecting groups; Azhayev et al, Tetrahedron 57, 4977-4986 (2001) for universal supports; Kozlov et al., Nucleosides, Nucleotides, and Nucleic Acids, 24 (5-7), 1037-1041 (2005) for improved synthesis of longer oligonucleotides through the use of large-pore CPG; and Damha et al., NAR, 18, 3813-3821 (1990) for improved derivitization).
Regardless of the type of synthesis, the resulting oligonucleotides may then form the smaller building blocks for longer oligonucleotides or gBlocks. As referenced earlier, the smaller oligonucleotides can be joined together using protocols known in the art, such as polymerase chain assembly (PCA), ligase chain reaction (LCR), and thermodynamically balanced inside-out synthesis (TBIO) (see Czar et al. Trends in Biotechnology, 27, 63-71 (2009)). In PCA oligonucleotides spanning the entire length of the desired longer product are annealed and extended in multiple cycles (typically about 55 cycles) to eventually achieve full-length product. LCR uses ligase enzyme to join two oligonucleotides that are both annealed to a third oligonucleotide. TBIO synthesis starts at the center of the desired product and is progressively extended in both directions by using overlapping oligonucleotides that are homologous to the forward strand at the 5′ end of the gene and against the reverse strand at the 3′ end of the gene.
Another method of synthesizing a larger double stranded DNA fragment or gBlock is to combine smaller oligonucleotides through top-strand PCR (TSP). In this method, a plurality of oligonucleotides span the entire length of a desired product and contain overlapping regions to the adjacent oligonucleotide(s). Amplification can be performed with universal forward and reverse primers, and through multiple cycles of amplification a full-length double stranded DNA product is formed. This product can then undergo optional error correction and further amplification that results in the desired double stranded DNA fragment (gBlock) end product.
In one method of TSP, the set of smaller oligonucleotides that will be combined to form the full-length desired product are between 40-200 bases long and overlap each other by at least about 15-20 bases. For practical purposes, the overlap region should be at a minimum long enough to ensure specific annealing of oligonucleotides and have a high enough melting temperature (Tm) to anneal at the reaction temperature employed. The overlap can extend to the point where a given oligonucleotide is completely overlapped by adjacent oligonucleotides. The amount of overlap does not seem to have any effect on the quality of the final product. The first and last oligonucleotide building block in the assembly should contain binding sites for forward and reverse amplification primers. In one embodiment, the terminal end sequence of the first and last oligonucleotide contain the same sequence of complementarity to allow for the use of universal primers.
Methods of mitigating synthesis errors are known in the art, and they optionally could be incorporated into methods of the present invention. The error correction methods include, but are not limited to, circularization methods wherein the properly assembled oligonucleotides are circularized while the other product remain linear and was enzymatically degraded (see Bang and Church, Nat. Methods, 5, 37-39 (2008)). The mismatches can be degraded using mismatch-cleaving endonucleases such as Surveyor Nuclease. Another error correction method utilizes MutS protein that binds to mismatches, thereby allowing the desired product to be separated (see Carr, P. A. et al. Nucleic Acids Res. 32, e162 (2004)).
Whether the oligonucleotides are combined through TSP or another form of assembly, the double stranded DNA gBlocks can then be combined with the bridging oligonucleotides of the present invention to produce larger DNA fragments that optionally contain one or more variable or repeat regions. The bridging oligonucleotides may contain fixed sequences to insert between gBlocks, or they may contain degenerate/mixed bases, or a combination thereof. In one embodiment the bridging oligonucleotide contains at least one mismatch within the overlap region in order to produce a large DNA fragment containing the bridge sequence and the adjacent gBlock sequences but for the substitution caused through the overlap mismatch.
The term “bridging oligonucleotide” refers to the single stranded oligonucleotide that contains ends at least partially complementary to the adjacent gBlocks. As illustrated in
In another embodiment, a single bridging oligonucleotide can combine more than two gBlocks. The bridging oligonucleotide can be long enough to overlap an entire sufficiently complementary strand of a first gBlock, wherein the bridging oligonucleotide is longer than the first gBlock to have 3′ and 5′ ends that can serve to hybridize to a second gBlock 3′ of the first gBlock and hybridize 5′ to a third gBlock, resulting in a new fragment that encodes for at least three gBlocks as well as the bridge sequences. In a further embodiment, the bridge can act as a constant variable, while the gBlock set can be diverse, such as a gBlock position using variable gBlocks for multiple promoters, or to prepare for multiple vectors.
The degenerate bases are a random mixture of multiple bases (also known as “mixed bases”), and for the purposes of this application can also refer to non-standard bases or spacers such as propanediol. For example, the degenerate bases may be an N mixture (a mixture of A, C, G and T bases), a K mixture (G and T bases), or an S mixture (G and C bases). Examples of non-standard bases include universal bases such as 3-nitropyrrole or 5-nitroindole.
The degenerate bases can be added for the purpose of increasing or reducing the GC content, or to construct a mutation library. In one embodiment a particular region of interest in a sequence is targeted to determine the effects of alternate bases on the expression of the encoded product. Only a relatively small amount of randomers inserted in the bridge could produce a large mutant library. Each N base would result in 4 different products. Each additional N base added by the bridging oligonucleotide would exponentially increase the library so that 2 N bases results in 16 combinations, 3 N bases results in 64, etc. By the time 18 N bases are inserted, the library contains over 68 billion different gene fragments. The cost of producing a library through the use of the methods of the invention is exponentially less expensive than through synthesizing each member of the library individually.
The bridging oligonucleotide will contain overlaps typically (but not limited to) 5-40 bases long on each side. The overlap is generally designed to create a bridging oligonucleotide/gBlock Tm of about 60-70° C. In one embodiment each overlap is about 15-25 bases long. Highly pure long single stranded oligonucleotides are commercially available up to 200 bases in length (e.g., Ultramer® oligonucleotides from Integrated DNA Technologies, Inc.), which would allow for 50 bases of overlap with each gBlock and up to 100 bases available for the bridge sequence. This allows for a large region (100 bases) to incorporate known sequence, degenerate bases, and combinations thereof. The degenerate bases may be consecutive, interrupted with known sequence, or concentrated in multiple areas along the bridge.
In another embodiment, degenerate or mismatch bases are incorporated into the adjacent gene block sequences through incorporating degenerate or mismatch bases within the overlap regions. In subsequent cycles of PCR to form a double-stranded product comprised of the gene block sequences and the bridge sequence, the mismatches will be incorporated into the longer product. The overlap regions can be designed to allow for adequate hybridization between the bridging oligonucleotide and the gBlock despite the mismatch.
In another embodiment, the bridging oligonucleotide is used to insert a sequence that is otherwise difficult to assemble or clone. The sequence may be difficult to assemble using PCR-based assembly methods using oligonucleotides such as TSP and is therefore added post-synthesis through the insertion of the sequence in the bridge portion of a bridging oligonucleotide.
In another embodiment, two or more bridging oligonucleotides can be combined with 3 or more gene blocks to assemble a DNA fragment or library resulting in combinations of one or more variable regions.
In another embodiment, a pool of individually synthesized bridging oligonucleotides can be pooled, wherein the two or more bridging oligonucleotides contain overlaps with the same two adjacent gene blocks but each contain a bridge sequence with degenerate region(s) located at successive positions along the length of the bridge sequence while keeping the rest of the bridge sequence constant (
In another embodiment, a pool of individually synthesized bridging oligonucleotides can be pooled, wherein the two or more bridging oligonucleotides contain non-random variation in the bridge sequence, such as specific codon or amino acid changes.
In another embodiment, one or more bridging oligonucleotides may consist exclusively of overlap sequences with the gene blocks, thereby combining the two gene blocks through PCR cycling without adding additional sequence between the two gene blocks.
Standard PCR methods well-known in the art, following the general scheme in
The gene blocks or libraries can then later be cloned through methods well-known in the art, such as isothermal assembly (e.g., Gibson et al. Science, 319, 1215-1220 (2008)); ligation-by-assembly or restriction cloning (e.g., Kodumal et al., Proc. Natl. Acad. Sci. U.S.A., 101, 15573-15578 (2004) and Viallalobos et al., BMC Bioinformatics, 7, 285 (2006)); TOPO TA cloning (Invitrogen/Life Tech.); blunt-end cloning; and homologous recombination (e.g., Larionov et al., Proc. Natl. Acad. Sci. U.S.A., 93, 491-496). The gene blocks can be cloned into many vectors known in the art, including but not limited to pUC57, pBluescriptII (Stratagene), pET27, Zero Blunt TOPO (Invitrogen), psiCHECK-2, pIDTSMART (Integrated DNA Technologies, Inc.), and pGEM T (Promega).
The gene blocks or libraries can be used in a variety of applications, not limited to but including protein expression (recombinant antibodies, novel fusion proteins, codon optimized short proteins, functional peptides—catalytic, regulatory, binding domains), microRNA genes, template for in vitro transcription (IVT), shRNA expression cassettes, regulatory sequence cassettes, micro-array ready cDNA, gene variants and SNPs, DNA vaccines, standards for quantitative PCR and other assays, and functional genomics (mutant libraries and unrestricted point mutations for protein mutagenesis, and deletion mutants).
One embodiment of the invention, a creation of a library in which multiple bridging oligonucleotides, each containing a degenerate region at successive positions, are pooled and assembled with double stranded DNA fragments to form a double stranded DNA walking library, could be used in a number of applications. This type of library is useful for introducing one amino acid change at a time along the sequence of interest, while keeping the other amino acids constant. This could be a useful tool in homologous recombination with gene editing technologies such as CRISPR.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
This example demonstrates the incorporation of low complexity sequences into a double stranded sequence through the use of a bridging oligonucleotide and double stranded DNA fragments (gBlocks). The method is useful for constructing DNA sequences that are difficult to assemble using conventional methods due to low sequence complexity, such as large repeat regions or homopolymeric runs.
As illustrated in
NNKGCATATATGGAATTGTCACGTTTGAGGTCTGATG
The assembled products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) at a bead:PCR volume ratio of 0.8:1, following manufacturer recommended conditions for washing and drying. The DNA was eluted using 45 μl of nuclease-free water and 5 μl of eluted DNA was added as the template into a second PCR reaction with the primers and the same PCR conditions used previously for assembly. These re-amplified PCR products were purified using AMPure XP magnetic beads as described previously and separated on a 2% agarose gel, stained with GelRed nucleic acid gel stain (Biotium), and visualized on a UV transilluminator. All of the re-amplified assemblies resulted in a single band of the expected size (
Error correction is an optional step that serves to decrease the number of mutations in the final construct. This was performed by first heating 100 ng of re-amplified assembly product in 20 ul of 1×HF buffer (New England Biolabs) to 95° C. and cooling slowly to form heteroduplex DNA where mutations are present. The heteroduplex DNA was treated with 1 μl Surveyor® Nuclease S (Integrated DNA Technologies) and 0.0125 units of exonuclease III (New England Biolabs) in 1×HF buffer and a final volume of 25 μl. The reaction was incubated at 42° C. for 1 hour.
After incubation, 5 μl of the error correction reaction was added as template in a PCR reaction using the same primers and reaction conditions as in the previous reactions. The post-error correction products were purified using AMPure XP magnetic beads using a bead:DNA volume ratio of 1:1 and separated on a 2% agarose gel and visualized as stated previously. All lanes contained the band of the expected size (
One pmole of each post-error correction product was subjected to Electrospray Mass Spectroscopy (ESI) analysis. The expected mass for each strand was obtained for all desired sequences and was the most prevalent species. Three examples are shown (
This example demonstrates the incorporation of 3 degenerate bases into a double stranded sequence through the use of a bridging oligonucleotide and double stranded DNA fragments to create a library of 32 DNA sequence variants. This type of library is useful for making single amino acid replacement libraries.
A double stranded DNA library containing a fixed region of degeneracy was created by incorporating NNK (N is the IUB code for A, G, C, T and K is the code for G or T) mixed base sites into the bridge sequence and assembling the bridging oligonucleotide between two double stranded DNA fragments. In this example the assembly was done using two gBlocks containing Illumina TruSeq P5 and P7 adapter sequences, which allowed for next generation sequencing analysis of the prevalence of mixed bases at each position in the final library.
P5 gBlock 1 (SEQ ID NO: 39) and P7AD002 gBlock 2 (SEQ ID NO: 40) were combined with the 1NNK bridge (SEQ ID NO: 41), which contained an internal NNK degenerate sequence flanked by 18 bases of sequence overlapping with each gBlock. The assembly PCR reaction contained equimolar 250 fmoles of each gBlock and bridging oligonucleotide, 200 nM primers (SEQ ID NO: 42 and 43), 0.02 U/μL of KOD Hot Start DNA polymerase, 1×KOD Buffer, 0.8 mM dNTPs and 1.5 mM MgSO4 in a 50 μl final volume. PCR cycling was performed using the following settings: (953:00−(950:20−610:10−700:20)×25 cycles. This resulted in the construction of the 1NNK gBlock library (SEQ ID NO: 44) with a complexity of 32 variants (42*21=32) and represents codons encoding all 20 standard amino acids and the stop codon TAG. The library was purified using AMPure XP magnetic beads at a bead:DNA volume ratio of 0.8:1, separated on a 2% agarose gel, and visualized as described in Example 1. A single band at the expected 355 base pair size was observed (
The 1NNK gBlock library was subjected to next-generation sequencing analysis on an Illumina MiSeq platform with a read length of 250×250 cycles. By only using overlapping paired end reads, the perfectly matched reads were used to determine the sequence and drastically lower the error rate from the sequencer.
This example demonstrates the contiguous incorporation of 18 degenerate bases into a double stranded sequence through the use of a bridging oligonucleotide and double stranded DNA fragments to create a library with more than 1 billion sequence variants. This type of library is useful for consecutive amino acid replacements.
A double stranded DNA library containing a highly complex region of degeneracy was created by assembling between two double stranded fragments a bridging oligonucleotide containing 6 tandem NNK degenerate regions. This allows the construction of a high complexity library [(42*21)6=1,073,741,824 variants]. The gBlock library was assembled using P5 gBlock 1 (SEQ ID NO: 39), P7AD009 gBlock 2 (SEQ ID NO: 45), 6NNK Bridge (SEQ ID NO: 46) and primers (SEQ ID NO: 42 and 43) under the same PCR conditions and purification described in example 2. This resulted in the construction of the 6NNK gBlock library (SEQ ID NO: 47).
The high complexity 6NNK gBlock library was subjected to next generation sequencing analysis on an Illumina MiSeq platform with a read length of 250×250 cycles.
This example demonstrates the incorporation of non-contiguous degenerate base positions into a double stranded sequence through the use of a bridging oligonucleotide and double stranded DNA fragments. This type of library is useful for introducing discrete islands of amino acid changes in between fixed sequence regions.
A double stranded DNA library containing non-contiguous degenerate base regions was created by assembling between two double stranded DNA fragments a bridging oligonucleotide containing one region of NNKNNK and two single NNK regions separated by 6 or 9 fixed DNA bases. GFP-A gBlock 1 (SEQ ID 048) and GFP-A gBlock 2 (SEQ ID 049) were combined with GFP-A Bridge (SEQ ID 050), which contained the regions of degeneracy flanked by overlap with each gBlock. The assembly PCR reaction contained equimolar 250 fmoles of each gBlock and bridging oligonucleotide, 200 nM primers (SEQ ID 051 and 052), 0.02 U/μL of KOD Hot Start DNA polymerase, 1×KOD Buffer, 0.8 mM dNTPs and 1.5 mM MgSO4 in a 50 μl final volume. PCR cycling was performed using the following settings: (953:00−(950:20−650:10−700:20)×25 cycles. This resulted in the construction of the GFP-A 444 bp library (SEQ ID 053).
The assembled library was diluted 100-fold in water and re-amplified (optional step) with just the terminal primers under the same PCR reaction and cycling conditions. The re-amplified library was separated on a 2% agarose gel and visualized as described in example 1. The full length product is 444 bp, and is indicated by a black star in
This example demonstrates the creation of a library in which multiple bridging oligonucleotides, each containing a degenerate region at successive positions, are pooled and assembled with double stranded DNA fragments to form a double stranded DNA walking library. This type of library is useful for introducing one amino acid change at a time along the sequence of interest, while keeping the other amino acids constant.
An example of the construction of a double stranded DNA library containing degenerate regions at successive positions along the sequence, while keeping the rest of the sequence constant, is illustrated in
The gBlock walking library product was purified with AMPure XP beads at a bead:DNA volume ratio of 0.8:1 and eluted in 25 μl water, followed by 100-fold dilution in water. The library was re-amplified (optional step) using 5 μl of the diluted library, 200 nM primers, and using the same PCR reaction conditions as in the previous step but with only 10 cycles of PCR. The libraries before and after 10 cycles of re-amplification were separated on a 2% agarose gel and visualized as described in example 1. The full length 408 bp product is present with or without re-amplification (
This example illustrates the detrimental effect of subjecting a double stranded DNA library containing a variable region to extensive PCR cycling during re-amplification.
Three different libraries were constructed using two gBlocks and one bridging oligonucleotide for each library assembly. The AD7 library (SEQ ID 073) was constructed using AD7 gBlock 1, AD7 gBlock 2, and AD7 Bridge (SEQ ID 070-072). The AD8 library (SEQ ID 077) was constructed using AD8 gBlock 1, AD8 gBlock 2, and AD8 Bridge (SEQ ID 074-076). The AD9 library (SEQ ID 081) was constructed using AD9 gBlock 1, AD9 gBlock 2, and AD9 Bridge (SEQ ID 078-080). The bridging oligonucleotide in each library contained 12 contiguous N mixed bases (equal mix of A, T, G, and C at each position) flanked by a region of overlap with each gBlock.
The library was assembled by combining equimolar amounts, 250 fmoles of gBlock1, gBlock 2, and bridging oligonucleotide for each library. The mixture was cycled at 95° C.3:00 (95° C.0:20+64° C.0:10+700:20)×25 cycles using 200 nM primers (Seq ID 068 and 069), 0.02 U/uL of KOD Hot Start DNA polymerase, 1×KOD buffer, 0.8 mM dNTP and 1.5 mM MgSO4 in a 50 μl final volume. The library product was purified with AMPure XP magnetic beads at a bead:DNA volume ratio of 0.8:1 and eluted in 45 μl water, followed by 100-fold dilution in nuclease-free water. Each library was re-amplified using 5 μl of the diluted library, 200 nM primers, and the same PCR reaction conditions as in the previous step but with either 10 or 20 cycles of PCR. The library products after re-amplification were separated on a 2% agarose gel and visualized as described in example 1 (
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This patent application claims priority to U.S. Provisional Patent Application No. 61/913,688 filed Dec. 9, 2013, the content of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61913688 | Dec 2013 | US |
