Synthesis of long nucleic acid sequences

Abstract

The invention provides methods for the synthesis of long oligonucleotides, genes and gene fragments. The methods include the manufacture of genes or gene fragments that can be then inserted into a variety of vectors.

Claims

1. A method of synthesizing a gene block of a desired sequence greater than 100 bases, the method comprising: a) providing a plurality of solid supports, wherein at least one solid support contains between 1-8% by weight controlled pore glass; b) synthesizing oligonucleotides on the solid supports, wherein the oligonucleotides comprise a set of oligonucleotides that are partially complementary to at least one other oligonucleotide in the set, and the set of oligonucleotides as a group contain the desired sequence of the gene block; c) detaching the oligonucleotides from the solid supports; d) combining the oligonucleotides under conditions to synthesize the gene block.

2. The method of claim 1 wherein at least one solid support contains 5-8% by weight controlled pore glass.

3. The method of claim 1 wherein the gene block is combined with additional gene blocks to form a longer gene block.

4. The method of claim 1 further comprising performing an error correction step after combining the oligonucleotides.

5. The method of claim 1 wherein the gene block is greater than 200 bases.

6. The method of claim 1 wherein the gene block is greater than 400 bases.

7. The method of claim 1 wherein the gene block is greater than 500 bases.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an illustration of top-strand PCR gene assembly. The upper portion, A, represents the first cycle of PCR, B the second cycle, and C is the desired full-length product.

(2) FIG. 2A is a gel showing the successful generation of the desired gene block assembled using six gene sub-blocks. FIG. 2B is a gel showing the successful generation of the desired gene block assembled using eight gene sub-blocks.

(3) FIGS. 3A and 3B graphically represent the amount of full length clones with no errors for each set of reactions.

(4) FIG. 4 depicts the 3 gene sub-block designs used to assemble the gene blocks according to Example 2.

(5) FIG. 5 is a collection of gels showing successful generation of the desired gene block assembled using varying gene sub-block designs using various sub-block concentrations.

(6) FIGS. 6A and 6B graphically represent the amount of full length clones with no errors for each set of reactions in Example 2.

(7) FIG. 7 is a gel showing a successful synthesis and assembly of a gene block as described in Example 3.

(8) FIG. 8 is a gel showing a successful synthesis and assembly of an 8 sub-block gene block and a 10 sub-block gene block as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

(9) 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 high fidelity oligonucleotide synthesis, the methods of use of high fidelity oligonucleotide synthesis in assembly/amplification methods such as top-strand PCR, and methods of gene assembly that yield a desired sequence, a gene block, through dilution of crude synthesized gene product, sequence verification and subsequent amplification.

(10) The term oligonucleotide, as used herein, refer 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, or double- and single-stranded oligonucleotides containing both RNA and DNA. 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.

(11) The terms raw material oligonucleotide and gene sub-blocks are used interchangeably in this application and refer 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.

(12) 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).

(13) 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.

(14) 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%). In one embodiment, the high-fidelity nucleotides are between 40-200 bases long. In a further embodiment the high-fidelity oligonucleotide is between 75-200 bases, and in a further embodiment 100-190 bases. High-fidelity oligonucleotides are available commercially, even at greater lengths (see Ultramer oligonucleotides from Integrated DNA Technologies, Inc.). Alternatively, a novel method of the present invention is the use of 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. The low-CPG membranes offer higher fidelity of array synthesis while still allowing for lower reagent use. Lower-CPG membranes are most practical when used to synthesize oligonucleotides greater than 50 bases, or further, greater than 75 bases.

(15) 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. Cem. 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).

(16) Regardless of the type of synthesis, the resulting oligonucleotides may then form the smaller building blocks for longer oligonucleotides. 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 oligos 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.

(17) One method of the present invention provides an alternative method of synthesis of the smaller oligonucleotides. In this method, top-strand PCR (TSP), a plurality of oligonucleotides span the entire length of a desired product and are partially complementary to the adjacent oligonucleotide(s) (see FIG. 1). Amplification is performed with universal forward and reverse primers, and through multiple cycles of amplification a full-length desired product is formed. This product can then undergo dilution, sequence screening, and further amplification that results in the desired gene block end product.

(18) In one method of TSP, the set of smaller oligonucleotides (gene sub-blocks) that will be combined to form the full-length desired product are between 40-200 bases long. In a further embodiment the oligonucleotide is between 75-200 bases, and in a further embodiment 100-190 bases. The gene sub-blocks overlap each other by at least 15-20 bases. For practical purposes, the overlap region should be at a minimum long enough to ensure specific annealing of gene sub-blocks and have a high enough melting temperature (T.sub.m) to anneal at the reaction temperature employed. The overlap can extend to the point where a given gene sub-block is completely overlapped by adjacent gene sub-blocks. 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 overlap regions contain the same sequence of complementarity to allow for the use of universal primers.

(19) Applicants have discovered that although intuitively more cycles of TSP (e.g., 30 cycles) would produce a greater percent of full-length molecules, surprisingly a greater percent of correct full-length molecules (i.e., assembled DNA strand having the desired sequence without error) is produced using a low number of cycles (about 15 cycles). After the initial TSP reaction of 15 cycles, the product can then be diluted 10-fold to 1000-fold, wherein the product is amplified again in 20-30 cycles of PCR to increase mass yield of the desired product.

(20) 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. 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)). Other mismatch nucleases include those in the CEL nuclease family (see Surveyor Nuclease, Transgenomics), or RES I (see Errase synthetic gene error correction kit, Novici). When error correction is used, the amount of remaining product may be small, and therefore a round of rescue PCR can be performed to amplify the desired product.

(21) Whether the gene sub-blocks are combined through TSP or another form of assembly, the full-length product is diluted, typically to 0.25 to 10 copies per reaction well, and then amplified to result in a large amount of homogeneous, desired double-stranded product (gene block). In a further embodiment, the synthesized oligonucleotides are diluted to 1-5 copies of the synthesized oligonucleotide per reaction well, and in a further embodiment the synthesized oligonucleotides are diluted to 2-4 copies per reaction well.

(22) The dilution and amplification steps replace the conventional, time-consuming, labor-intensive in vivo cloning procedures traditionally employed, which are well known in the art. Some dilution methods are known in the art (see Yehezkel et al., Nucleic Acids Research, vol. 36, no. 17, e107 (2008)), but they have been inefficient. The goal of dilution PCR is to dilute the initial product, which is a mixture of desired perfect product and undesired imperfect products, into reaction wells to a dilution that best assures that an adequate amount of reaction wells contain the desired product. If the mixture is too dilute then there are undesired empty wells, and if the mixture is not diluted enough then too many wells contain multiple template molecules (product of desired sequence plus defective product containing an undesired sequence). Since DNA sequencing is a significant portion of gene synthesis cost, typical dilutions would normally be less than one molecule per well, and even as low as 0.2 molecules per well to ensure clonality.

(23) In the methods of the present invention, dilution conditions can be used that allow for as much as 1-5 copies of oligonucleotides/well to be present. Because the fidelity of the gene sub-blocks made using the methods of the present invention is so high, the product being diluted is weighted heavily to contain a high percentage of correct sequence material. Therefore, if a given well has 4 molecules, there is still a high likelihood that the amplified product will be of the correct sequence. Diluting to an average copy of 1-5, or more preferably 2-4, reduces the number of empty wells and increases the efficiency of the manufacturing process.

(24) The resulting product after dilution and amplification is then directly sequence verified without the traditional need to first clone the product into a plasmid vector and expand in a bacterial host. The final product is a desired, sequence verified gene block. The gene blocks 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).

(25) The above methods can be re-ordered or altered, or further steps can be incorporated to optimize the end product, particularly if the known end-product is shorter or longer. For example, in one embodiment where the desired end product is a gene block smaller than 500 bases, the initial steps would be to synthesize the gene sub-blocks, perform PCR (e.g., TSP), then dilute and amplify. The resulting product is then treated with error correction and then undergoes amplification, such as PCR. Optionally, the product undergoes a second dilution step and amplification. The end product then is re-amplified with sequence-specific primers if there is a need to remove universal sequences inserted for use in earlier amplification steps.

(26) For longer desired products (i.e., greater than 500 bases), a number of options are available to manufacture the longer end product. In one embodiment, longer TSP-assembled starting material is used. In another embodiment, two or more smaller products are used, and those products undergo isothermal assembly. Those products could be combined with other products to make even longer gene blocks. In another embodiment, two or more TSP-assembled products that undergo isothermal assembly. In another embodiment, a set of oligonucleotides of about 60 bases in length, wherein the oligonucleotides overlap with adjacent oligonucleotides to cover a 1-2 kb sequence length, are combined and undergo isothermal assembly.

(27) The gene blocks 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 peptidescatalytic, 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). The ease of synthesizing large genes or gene segments using gene blocks allows for the synthesis of a set of large segments/genes wherein one or more gene blocks remain constant while one or more gene blocks varies.

(28) The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

(29) This example illustrates an assembly of gene sub-blocks into a product containing desired gene blocks.

(30) A 935 bp gene block comprising 6 synthetic oligonucleotide subunits and a 1155 bp gene block comprising 8 synthetic oligonucleotide subunits were assembled. The 8-oligonucleotide gene block is an extension of the smaller 935 bp 6-oligonucleotide gene block sequence having an additional 220 bp at the 3-end. Therefore both gene blocks use the same 6 component oligonucleotides in assembly, and the 8-oligonucleotide gene block also includes oligonucleotides 7 and 8. The sequences of the gene blocks, the component oligonucleotides, the universal forward primer, the universal reverse primer (8-block) and gene specific reverse primers (6-block). Gene blocks were assembled using component oligonucleotides having an unmodified 3-end, having 6 additional non-templated T bases added to the 3-end of each oligonucleotide (T-blocked), having 8 additional non-templated T bases plus a terminal C3 spacer (propanediol) (T+C3-blocked), or having a terminal C3 spacer (propanediol) added to the end of each oligonucleotide. The different end-blocked versions of assembly will test whether extension from the 3-end of component oligonucleotides is necessary during TSP. The following sequences were used:

(31) TABLE-US-00001 935baseGeneBlock(SEQIDNo.1): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCG ATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGG CAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGC GGCTTCCATCAAAATTTCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGC AGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATTTT CCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGCGT GAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAG ATTCTAAAGACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGT GTACGCTTGCGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAA GCTGGGGATCCTCTAGAGGTTGAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGT GTTCTCTATCGCCACCAATGCGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAA CCGGGGGCCTCTGTGAAAGTCTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGC GTCAAGCCCCGGGTCAAGGTCTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGT 1155baseGeneBlock(SEQIDNo.2): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCG ATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGG CAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGC GGCTTCCATCAAAATTTCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGC AGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATTTT CCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGCGT GAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAG ATTCTAAAGACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGT GTACGCTTGCGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAA GCTGGGGATCCTCTAGAGGTTGAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGT GTTCTCTATCGCCACCAATGCGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAA CCGGGGGCCTCTGTGAAAGTCTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGC GTCAAGCCCCGGGTCAAGGTCTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGTACCAACTATGCGCA GAAATTCCAGGGTCGCGTCACGATGACTCGCGACACGTCAATTAGTACCGCGTACATGGAGTTATCGCGTTTA CGTAGTGACGACACCGCCGTATACTACTGTGCGCGTGCTCAGAAACGCGGCCGTTCTGAATGGGCGTACGCAC ATTGGGGTCAAGGCACCCTGGTGACCGTGAGTAGTGGATCGACGAGAGCAGCGCGACTGG Componentoligonucleotide1(SEQIDNo.3): CGACACTGCTCGATCCGCTCGCACCAAATCCAGATGACACAGAGTCCGTCCTCGCTTTCTGCGTCCCTGGGCG ATCGTGTAACCATTACATGTCAGGCTAGTCGCGGCATCGGAAAAGACTTAAATTGGTACCAGCAGAAAGCGGG CAAAGCCCCTAAACTGCTGGTGAGCGATGCCAGCACATTGGAG Componentoligonucleotide2(SEQIDNo.4): CTGGTGAGCGATGCCAGCACATTGGAGGGCGGCGTTCCGTCACGTTTCAGTGGTAGCGGCTTCCATCAAAATT TCAGCTTAACCATCTCCAGTCTGCAGGCCGAGGATGTGGCTACCTATTTCTGCCAGCAGTATGAAACTTTCGG CCAGGGAACCAAAGTC Componentoligonucleotide3(SEQIDNo.5): GCAGTATGAAACTTTCGGCCAGGGAACCAAAGTCGATATTAAAAGGTCGACGGTCGCGCCGAGCGTGTTTATT TTCCCGCCGTCTGATGAACAGCTGAAATCAGGCACCGCATCGGTGGTTTGCCTGCTGAACAATTTTTATCCGC GTGAAGCGAAAGTTCAGTGGAAAGTGGATAACGCCCTGCAGAG Componentoligonucleotide4(SEQIDNo.6): GTTCAGTGGAAAGTGGATAACGCCCTGCAGAGCGGTAATTCGCAAGAAAGCGTCACCGAACAAGATTCTAAAG ACAGTACGTACTCCCTGAGCTCTACCCTGACGCTGTCAAAAGCAGATTACGAAAAACATAAAGTGTACGCTTG CGAAGTTACCCACCAAGGCCTGAGTTCCCCGGTTACGAAATCC Componentoligonucleotide5(SEQIDNo.7): CCAAGGCCTGAGTTCCCCGGTTACGAAATCCTTCAACCGTGGCGAATGTTAAGCTGGGGATCCTCTAGAGGTT GAGGTGATTTTATGAAGAAAAACATTGCGTTTCTGCTGGCGAGCATGTTTGTGTTCTCTATCGCCACCAATGC GTATGCCCTCGAGCAAGTGCAATTGGTC Componentoligonucleotide6(SEQIDNo.8): CGTATGCCCTCGAGCAAGTGCAATTGGTCCAGTCGGGCGCGGAAGTTAAGAAACCGGGGGCCTCTGTGAAAGT CTCCTGCAAAGCCTCTGGTTATACGTTTACAGGCTACTATATGCACTGGGTGCGTCAAGCCCCGGGTCAAGGT CTGGAGTGGATGGGTTGGATTAACCCGAACTCCGGTGGT Componentoligonucleotide7(SEQIDNo.9): GGGTTGGATTAACCCGAACTCCGGTGGTACCAACTATGCGCAGAAATTCCAGGGTCGCGTCACGATGACTCGC GACACGTCAATTAGTACCGCGTACATGGAGTTATCGCGTTTACGTAGTG Componentoligonucleotide8(SEQIDNo.10): TACCGCGTACATGGAGTTATCGCGTTTACGTAGTGACGACACCGCCGTATACTACTGTGCGCGTGCTCAGAAA CGCGGCCGTTCTGAATGGGCGTACGCACATTGGGGTCAAGGCACCCTGGTGACCGTGAGTAGTGGATCGACGA GAGCAGCGCGACTGG UniversalForprimer5-phos(SEQIDNo.11): /5Phos/CGACACTGCTCGATCCGCTCGCACC UniversalRevprimer5-phos(SEQIDNo.12): /5Phos/CCAGTCGCGCTGCTCTCGTCGATCC GeneSpecificRevprimer(SEQIDNo.13): /5Phos/ACCACCGGAGTTCGGGTTAATCCAACC

(32) The component oligonucleotides were assembled by TSP using the following reaction mixture and conditions:

(33) 50/100 nM component oligonucleotides 1-6/8

(34) 50/100 nM forward primer

(35) 200 nM reverse primer

(36) 0.02 U/uL KOD Hot-Start DNA polymerase (Novagen)

(37) 1 buffer for KOD Hot Start DNA polymerase (Novagen)

(38) 1.5 mM MgSO.sub.4

(39) 0.8 mM dNTPs (0.2 mM each)

(40) Cycling conditions: 95 C..sup.3:00 (95 C..sup.0:2070 C..sup.0:30)15, 20, 25, or 30 cycles

(41) Additionally, several sets of otherwise identical component oligonucleotides were used in TSP assembly wherein the 3 ends are either unblocked or blocked with a 6-residue poly-T; a C3 spacer; or a 8-residue poly-T plus a C3 spacer. After the TSP cycles, the resulting products were diluted 100-fold in water, and then underwent a further step of PCR containing 200 nM each of the universal forward primer and 200 nM of the universal reverse primer (8-block) or gene specific reverse primer (6-block) (cycling conditions: 95 C..sup.3:00 (95 C..sup.0:2070 C..sup.0:30)30 cycles). The resulting gene block was run on a 1.2% agarose gel at 100V for 1 hour 30 minutes.

(42) FIG. 2A is a picture of the resulting gel of the 935-base 6-component oligonucleotides gene block, and FIG. 2B is the resulting gel for the 1155 base 8-component oligonucleotides gene block. Notably, at 15 TSP cycles the desired product is present, particularly using the unblocked component oligonucleotides. The 50 nM 6-block gene blocks were blunt cloned into a pUC19 vector and sequence verified using Sanger sequencing using a 3730xl sequencer (Life Technologies-Applied Biosystems). FIG. 3A graphically represents the percent of full length clones with no errors for each set of reactions. FIG. 3B represents the percent of all clones with no errors after sequencing additional clones generated using unblocked component oligonucleotides. Although all conditions produce full-length clones with no errors, surprisingly the low cycle number unblocked component oligonucleotides was the most robust and yielded the highest percentage of correct final products.

EXAMPLE 2

(43) This example demonstrates that various lengths, concentrations and numbers of component oligonucleotides and varying component oligonucleotide overlap conditions can be used to successfully produce full length gene blocks.

(44) FIG. 4 illustrates the three overlap designs used in this example. Design 1 uses component oligonucleotides that are 115-185 nucleotides in length. Design 2 uses shorter component oligonucleotides, between 54-125 nucleotides. Design 3 uses 100 nucleotidelong component oligonucleotides (the last oligonucleotide may be shorter or longer than 100 nucleotides depending on final gene block length), and employs a complete overlap design wherein the adjacent oligonucleotides completely overlap each non-terminal component.

(45) The following are the sequences of the desired gene blocks and component oligonucleotides. The same universal For and Rev primers from Example 1 (SEQ ID Nos. 11-12) were used.

(46) TABLE-US-00002 GeneBlockA(SEQIDNo.14): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTAT TGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATC CTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGT GATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCC TACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGG CCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTC TAAGGGGATCGACGAGAGCAGCGCGACTGG GeneBlockB(SEQIDNo.15): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGAC CTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGA CATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTA CCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAG GAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCC CAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCAT GGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockC(SEQIDNo.16): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGT CGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGT GGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCC AATGCCGCTGCCCCCCAGCGCATTGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACC CTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGG GACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCC GGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockADesign1Componentoligonucleotide1(SEQIDNo.17): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTAT TGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATC CTCCAAGCCGGACTGCCGGCAAATGCCTCCACAG GeneBlockADesign1Componentoligonucleotide2(SEQIDNo.18): GGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAG CCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAAG GTTCTCAAGGTGAGGACTTTCTGAATCTAAAGG GeneBlockADesign1Componentoligonucleotide3(SEQIDNo.19): CCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGGCC ACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTA AGGGGATCGACGAGAGCAGCGCGACTGG GeneBlockADesign2Componentoligonucleotide1(SEQIDNo.20): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGG GeneBlockADesign2Componentoligonucleotide2(SEQIDNo.21): GGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTC CTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCC GeneBlockADesign2Componentoligonucleotide3(SEQIDNo.22): CGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAG TTTGTCTGCAAGGTTTACAGTGATG GeneBlockADesign2Componentoligonucleotide4(SEQIDNo.23): GGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAG AACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACC GeneBlockADesign2Componentoligonucleotide5(SEQIDNo.24): GCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTC TCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAG GeneBlockADesign2Componentoligonucleotide6(SEQIDNo.25): GGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAG AGTGTTCTAAGGGGATCGACGAGAGCAGCGCGACTGG GeneBlockADesign3Componentoligonucleotide1(SEQIDNo.26): CGACACTGCTCGATCCGCTCGCACCTTTCTGGCATGAGGTCACTGACAGCCCTCTGGACAACACAGCTTATTTAT TGGTCTCTCATTCTCCCATCCCCAC GeneBlockADesign3Componentoligonucleotide2(SEQIDNo.27): CCTCTGGACAACACAGCTTATTTATTGGTCTCTCATTCTCCCATCCCCACTCCTCCTTTCTTCCCTCTCTCCACC AGAGCGATGGCGTCACCGGCCCATC GeneBlockADesign3Componentoligonucleotide3(SEQIDNo.28): TCCTCCTTTCTTCCCTCTCTCCACCAGAGCGATGGCGTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATG CCTCCACAGTGGTCGGAGGAGACGT GeneBlockADesign3Componentoligonucleotide4(SEQIDNo.29): CTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGTCGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGT GATGCCCAGCCCCACATCCAGTGGA GeneBlockADesign3Componentoligonucleotide5(SEQIDNo.30): AGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTGGATCAAGCACGTGGAAAAGAACGGCAG TAAATACGGGCCCGACGGGCTGCCC GeneBlockADesign3Componentoligonucleotide6(SEQIDNo.31): TCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAAGGTTCTCAAGGTGAGGA CTTTCTGAATCTAAAGGTACCCACA GeneBlockADesign3Componentoligonucleotide7(SEQIDNo.32): TACCTCAAGGTTCTCAAGGTGAGGACTTTCTGAATCTAAAGGTACCCACAACTGGGGTCTCCTTCATGGGTTTGG CCACAGGTTCTTTGATTTCCTGTTG GeneBlockADesign3Componentoligonucleotide8(SEQIDNo.33): ACTGGGGTCTCCTTCATGGGTTTGGCCACAGGTTCTTTGATTTCCTGTTGGAGTTGAGAGAGGATGATTCTCTTT TTTGACTAGCCAGCAGAGAGTGTTC GeneBlockADesign3Componentoligonucleotide9(SEQIDNo.34): GAGTTGAGAGAGGATGATTCTCTTTTTTGACTAGCCAGCAGAGAGTGTTCTAAGGGGATCGACGAGAGCAGCGCG ACTGG GeneBlockBDesign1Componentoligonucleotide1(SEQIDNo.35): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGAC CTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAG GeneBlockBDesign1Componentoligonucleotide2(SEQIDNo.36): GTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAA AGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGAT GAACCCACTCTACAGCTGGAGGCTGTAAATCCC GeneBlockBDesign1Componentoligonucleotide3(SEQIDNo.37): GAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTC AACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATG GAAGCTG GeneBlockBDesign1Componentoligonucleotide4(SEQIDNo.38): CTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGT CACCAAATTCTACATGCAATTTGTGTCTTCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockBDesign2Componentoligonucleotide1(SEQIDNo.39): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAG GeneBlockBDesign2Componentoligonucleotide2(SEQIDNo.40): CGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGA TACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCC GeneBlockBDesign2Componentoligonucleotide3(SEQIDNo.41): ATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCT TGGGCCTCAAGG GeneBlockBDesign2Componentoligonucleotide4(SEQIDNo.42): ACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACA GCTGGAGGCTGTAAATCCC GeneBlockBDesign2Componentoligonucleotide5(SEQIDNo.43): GAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTC AACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTTCCC GeneBlockBDesign2Componentoligonucleotide6(SEQIDNo.44): AACCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAG CCTCACCAATATGCCTGACGAAGGC GeneBlockBDesign2Componentoligonucleotide7(SEQIDNo.45): GTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCCGGATCG ACGAGAGCAGCGCGACTGG GeneBlockBDesign3Componentoligonucleotide1(SEQIDNo.46): CGACACTGCTCGATCCGCTCGCACCGCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACCAGAAGAC CTTCTATCTGAGGAACAACCAACTA GeneBlockBDesign3Componentoligonucleotide2(SEQIDNo.47): GAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAA ATGTCAATTTAGAAGAAAAGTTCGA GeneBlockBDesign3Componentoligonucleotide3(SEQIDNo.48): AGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCGACATGTCCTTTGTAGAAGGACATGA AAGTAATGACAAAATACCTGTGGCC GeneBlockBDesign3Componentoligonucleotide4(SEQIDNo.49): ACATGTCCTTTGTAGAAGGACATGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGT ACCTGTCCTGCGTGTTGAAAGATGA GeneBlockBDesign3Componentoligonucleotide5(SEQIDNo.50): CTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATGAACCCACTCTACAGCTGGAGGC TGTAAATCCCAAAAATTACCCAAAG GeneBlockBDesign3Componentoligonucleotide6(SEQIDNo.51): ATGAACCCACTCTACAGCTGGAGGCTGTAAATCCCAAAAATTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCT TCAACAAGATAGATTCAGGCCCAAC GeneBlockBDesign3Componentoligonucleotide7(SEQIDNo.52): GAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGATTCAGGCCCAACCACATCATTTGAGTCTGCCCAGTT CCCCAACTGGTTCCTCTGCACAGCG GeneBlockBDesign3Componentoligonucleotide8(SEQIDNo.53): CCACATCATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCC TCACCAATATGCCTGACGAAGGCGT GeneBlockBDesign3Componentoligonucleotide9(SEQIDNo.54): GATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCA ATTTGTGTCTTCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockCDesign1Componentoligonucleotide1(SEQIDNo.55): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGT CGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGA GeneBlockCDesign1Componentoligonucleotide2(SEQIDNo.56): GGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCG GTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCAT CGCCATCGCCATTG GeneBlockCDesign1Componentoligonucleotide3(SEQIDNo.57): CATTGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCG CGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTG GeneBlockCDesign1Componentoligonucleotide4(SEQIDNo.58): GTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGA GCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGA CTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockCDesign2Componentoligonucleotide1(SEQIDNo.59): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCC GeneBlockCDesign2Componentoligonucleotide2(SEQIDNo.60): GCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGG CTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGG GeneBlockCDesign2Componentoligonucleotide3(SEQIDNo.61): TGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGAC GCAATTCTGCTG GeneBlockCDesign2Componentoligonucleotide4(SEQIDNo.62): GTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCAT CGCCATCGCCATTGCTG GeneBlockCDesign2Componentoligonucleotide5(SEQIDNo.63): CACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGG ACGAGCTGTACGCCGAGTGGTCGGAGGTC GeneBlockCDesign2Componentoligonucleotide6(SEQIDNo.64): GCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGG CGAGCAGCC GeneBlockCDesign2Componentoligonucleotide7(SEQIDNo.65): CCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACT TCGTGGCCGAGGAGCAGGACTAAGG GeneBlockCDesign2Componentoligonucleotide8(SEQIDNo.66): CACTTCGTGGCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG GeneBlockCDesign3Componentoligonucleotide1(SEQIDNo.67): CGACACTGCTCGATCCGCTCGCACCCATATGGCCAAGCTGACCAGCGCCGTTCCGGTGCTCACCGCGCGCGACGT CGCCGGAGCGGTCGAGTTCTGGACC GeneBlockCDesign3Componentoligonucleotide2(SEQIDNo.68): TTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACT TCGTGGAGGACGACTTCGCCGGTGT GeneBlockCDesign3Componentoligonucleotide3(SEQIDNo.69): CGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTT CATCAGCGCGGTCCAGGACCAGGTA GeneBlockCDesign3Componentoligonucleotide4(SEQIDNo.70): TGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTAAGACGCAATTCTGCTGTGCACGTGC CAATGCCGCTGCCCCCCAGCGCATT GeneBlockCDesign3Componentoligonucleotide5(SEQIDNo.71): AAGACGCAATTCTGCTGTGCACGTGCCAATGCCGCTGCCCCCCAGCGCATTGGCTCACCATCGCCATCGCCATTG CTGCTGCAGGTGGTGCCGGACAACA GeneBlockCDesign3Componentoligonucleotide6(SEQIDNo.72): TGGCTCACCATCGCCATCGCCATTGCTGCTGCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGG CCTGGACGAGCTGTACGCCGAGTGG GeneBlockCDesign3Componentoligonucleotide7(SEQIDNo.73): ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTC CGGGACGCCTCCGGGCCGGCCATGA GeneBlockCDesign3Componentoligonucleotide8(SEQIDNo.74): GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGG GCGGGAGTTCGCCCTGCGCGACCCG GeneBlockCDesign3Componentoligonucleotide9(SEQIDNo.75): ACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTG GCCGAGGAGCAGGACTAAGGATCCGGATCGACGAGAGCAGCGCGACTGG

(47) The Component oligonucleotides were pooled, assembled using TSP, diluted and subsequently amplified with PCR as was done previously in Example 1. As in Example 1, the resulting product was run on gels (see FIG. 5) and DNA sequence of the amplification product was determined for gene blocks generated using 25 nM or 100 nM of each component oligonucleotide and run for 15 TSP cycles (see FIGS. 6A and 6B). As the gels and graphs indicate, each design method, at varying cycles, produces the desired gene block product. While any of the assembly methods outlined above can be used with the method of the invention, use of the longer component oligonucleotides with less overlap is preferred to simplify design and automation as well as lowering materials cost. Thus fewer, longer component oligonucleotides can be utilized under short cycling conditions to assemble the desired gene block.

EXAMPLE 3

(48) This example demonstrates the synthesis of a gene block using TSP, followed by dilution and subsequent amplification of the desired sequence gene block from a heterogeneous assembly mixture.

(49) TABLE-US-00003 Example3GeneBlock(SEQIDNo.76): ACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCAT CTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAG CCCCCAAACTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCT GGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTCTTCACTGAC AGACAGAAGCCATCGCATATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGG TCACTCTGTTCCCGCCCTCGAG Example3Componentoligonucleotides: SEQIDNo.77: CGACACTGCTCGATCCGCTCGCACCACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTG GGTCTCCTGGACAGTCGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCC TGGTACCAACAGCACCCAGGCAAAG SEQIDNo.78: CTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGG TTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC GAG SEQIDNo.79: CTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCG GCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCGAGG GATCGACGAGAGCAGCGCGACTGG Forwardprimer(SEQIDNo.80): /5Phos/ACCGGTTCCTGGGCC(59.3 Tm) ReversePrimer(SEQIDNo.81): /5Phos/CTCGAGGGCGGGAACAG(60.1 Tm)

(50) There is a 29-base overlap between the first and second component oligonucleotides (70.1 Tm), and a 25-base overlap between the second and third component oligonucleotides (69.4 Tm). TSP assembly was performed on a disposable tip Janus robot, the product was then diluted (5 l of the final product of the TSP cycling diluted into 145 l of water), and the diluted aliquot was further amplified by PCR using the indicated terminal Forward and Reverse primers.

(51) TSP Reaction Mixture

(52) The TSP reaction was set up in a final reaction volume of 25 L. Each oligonucleotide (including the forward and reverse universal primers) were at a final concentration of 140 nM in a 1KOD DNA polymerase buffer. The reaction contained a final concentration of 0.8 mM dNTPs, 1.5 mM MgSO.sub.4 and 0.5 U of KOD DNA polymerase (Novagen). The cycling parameters were: 95.sup.3:00(95.sup.0:1570.sup.0:30)30. After the initial cycling, the reaction was diluted 1:5 and was reamplified with the addition of fresh forward and reverse universal primers, MgSO.sub.4, and dNTPs under the same cycling conditions.

(53) The amplification products were separated by agarose gel electrophoresis and visualized by fluorescent dye staining to verify the length of the assembled gene block. Sample cleanup was performed (QIAquick PCR Purification Kit, Qiagen) and the product was quantified via UV absorbance (Abs=0.034, Conc.=22.1 ng/uL). Serial dilution was performed (Janus DT robot) with IDTE buffer 8.0 pH w/tRNA at a conc. 0.1 mg/ml for a final dilution of 0.3 copies/5 L.

(54) Serial Dilutions:

(55) 1=5 uL of Sample, 145 L of diluent, MIX

(56) 2=5 uL of first dilution, 71 L of diluent, MIX

(57) 3=15 uL of 2.sup.nd dilution, 145 L of diluent, MIX

(58) 4=15 uL of 3.sup.rd dilution, 145 L of diluent, MIX

(59) 5=15 uL of 4.sup.th dilution, 145 L of diluent, MIX

(60) 6=15 uL of 5.sup.th dilution, 145 L of diluent, MIX

(61) 7=15 uL of 6.sup.th dilution, 145 L of diluent, MIX

(62) 8=15 uL of 7.sup.th dilution, 145 L of diluent, MIX

(63) 9=15 uL of 8.sup.th dilution, 145 L of diluent, MIX

(64) 10=15 uL of 9.sup.th dilution, 145 L of diluent, MIX

(65) 11=15 uL of 10.sup.th dilution, 145 L of diluent, MIX

(66) Dilution PCR: Plates containing 1KOD buffer, 0.25 U KOD DNA polymerase, 1.5 mM MgSO.sub.4, 300 nM forward and reverse universal primers, and 0.6 EvaGreen, 5 L of the final dilution of the assembled gene block, all in a final volume of 25 L. The cycling conditions were 95.sup.2:00(95.sup.0:2070.sup.1:00)45.

(67) Wells containing a positive fluorescent signal were diluted 1:16 in water. The diluted amplified product was then sequence verified using standard Sanger based sequencing on a 3730XL DNA sequencer. One L of the diluted clonally amplified product was further amplified by PCR using gene specific terminal primers under the following conditions; 1KOD buffer, 0.5 U KOD DNA polymerase, 1.5 mM MgSO.sub.4, 0.8 mM dNTPs, and 200 nM primers. The cycling conditions were 95.sup.3:00(95.sup.0:1560.sup.0:1570.sup.0:30)30 cycles.

(68) The TSP generated gene block and dilution-amplified gene block were separated using agarose gel electrophoresis and visualized by fluorescent dye staining. A 1 kb marker size ladder (Axygen) was included (see FIG. 7). The size markers are shown for the four smallest standards. The expected product is 442 bp, and products of this size were visualized on the gel.

EXAMPLE 4

(69) The present example demonstrates an alternate protocol wherein the TSP cycling conditions are modified to allow for longer extension times to make longer gene blocks. The longer cycling conditions significantly improve the resulting desired product.

(70) The 8 sub-block gene block (1155 bases, SEQ ID No. 2) from Example 1 was synthesized, as well as a 10 sub-block gene block (1308 bases, SEQ ID No. 82). The 10 sub-blocks are SEQ ID Nos. 83-92.

(71) The component oligonucleotides were assembled by TSP using the following reaction mixture and conditions:

(72) 50 nM component oligonucleotides

(73) 50 nM forward primer

(74) 200 nM reverse primer

(75) 0.02 U/uL KOD Hot-Start DNA polymerase (Novagen)

(76) 1 buffer for KOD Hot Start DNA polymerase (Novagen)

(77) 1.5 mM MgSO.sub.4

(78) 0.8 mM dNTPs (0.2 mM each)

(79) Cycling conditions: 95 C..sup.3:00 (95 C..sup.0:2070 C..sup.0:40, 0:50, or 1:00)40 or 50 cycles

(80) After the TSP cycles, the resulting products were diluted 100-fold in water, and then underwent a further step of PCR containing 200 nM each of the universal forward primer and 200 nM of the universal reverse primer (cycling conditions: 95 C..sup.3:00 (95 C..sup.0:20-70 C..sup.0:40)30 cycles). The resulting gene block was run on a 1% agarose gel at 100V for 1 hour 15 minutes (see FIG. 8). The expected products were 1155 bp and 1308 bp respectively, and products of these sizes were visualized on the gel.

(81) TABLE-US-00004 SEQIDNo.82:10sub-blockgeneblock CGACACTGCTCGATCCGCTCGCACCCCGCCTTGTTTAACTTTAAGAAGGAGCCCTTCCCCATGACAAGAACAA GTTTGCCTTTTCCAGACGGTTTCCTGTGGGGCGCAAGCACGGCGGCTCACCAGATTGAAGGTAATAATGTAAA TAGTGATTGGTGGAGAAAAGAACATGACCCTGCTGCAAATATTGCAGAACCATCTTTGGATGCCTGTGACTCA TATCACCGCTGGGAACAAGATATGGACCTGTTAGCAGAACTGGGCTTTACCGATTACCGCTTCTCCGTTGAAT GGGCCCGTATTGAACCTGTGCCAGGTACATTTTCGCATGCTGAAACGGCACACTATCGTAGAATGGTTGATGG TGCTTTGGCAAGAGGCCTGCGCCCAATGGTCACCCTGCATCACTTTACTGTACCGCAGTGGTTCGAAGATTTG GGTGGCTGGACAGCCGATGGTGCCGCGGACCTGTTTGCACGTTACGTCGAACATTGTGCTCCGATTATCGGTA AAGATGTTAGACACGTGTGCACGATTAATGAACCTAACATGATCGCCGTAATGGCGGGCTTAGCTAAGACAGG CGATCAAGGTTTCCCACCGGCGGGTTTGCCTACGCCTGACGAAGAAACCACTCATGCTGTTATTGCTGCACAT CACGCCGCGGTCAAAGCAGTACGTGCCATTGATCCGGACATCCAGGTCGGCTGGACCATCGCTAATCAAGTAT ATCAGGCATTACCTGGTGCCGAAGATGTTACTGCTGCATATCGTTACCCAAGAGAAGACGTGTTCATTGAAGC CGCTCGTGGCGATGACTGGATCGGCGTGCAATCTTACACACGCACGAAGATTGGTGCGGATGGCCCAATCCCG GCGCCTGAAGACGCTGAACGCACCCTGACTCAGTGGGAATATTACCCAGCTGCTGTTGGTCATGCTCTGCGTC ACACAGCGGATGTCGCTGGCCCAGACATGCCGTTAATTGTAACCGAAAACGGTATCGCCACTGCGGATGACGC ACGCCGTGTGGATTATTACACTGGTGCACTGGAAGCCGTTTCAGCCGCGTTAGAAGATGGTGTGAATATTCAT GGCTATCTGGCGTGGAGCGCTTTGGATAACTATGAATGGGGTAGTTACAAACCGACTTTTGGCCTGATCGCAG TTGATCCTGTGACATTCGAAAGAACGGCCAAGCCGTCAGCAGTGTGGTTAGGTGAAATGGGTAGAACAAGACA GTTGCCAAGAGCGGAACGCGGGAAGGGTGGGCGCGCCGACCCGGATCGACGAGAGCAGCGCGACTGG Sub-blocksequences: SEQIDNo.83: CGACACTGCTCGATCCGCTCGCACCCCGCCTTGTTTAACTTTAAGAAGGAGCCCTTCCCCATGACAAGAACAA GTTTGCCTTTTCCAGACGGTTTCCTGTGGGGCGCAAGCACGGCGGCTCACCAGATTGAAGGTAATAATGTAAA TAGTGATTGGTGGAG SEQIDNo.84: GCTCACCAGATTGAAGGTAATAATGTAAATAGTGATTGGTGGAGAAAAGAACATGACCCTGCTGCAAATATTG CAGAACCATCTTTGGATGCCTGTGACTCATATCACCGCTGGGAACAAGATATGGACCTGTTAGCAGAACTGGG CTTTACCGATTACCGCTTCTCCGTTG SEQIDNo.85: GAACTGGGCTTTACCGATTACCGCTTCTCCGTTGAATGGGCCCGTATTGAACCTGTGCCAGGTACATTTTCGC ATGCTGAAACGGCACACTATCGTAGAATGGTTGATGGTGCTTTGGCAAGAGGCCTGCGCCCAATG SEQIDNo.86: CTTTGGCAAGAGGCCTGCGCCCAATGGTCACCCTGCATCACTTTACTGTACCGCAGTGGTTCGAAGATTTGGG TGGCTGGACAGCCGATGGTGCCGCGGACCTGTTTGCACGTTACGTCGAACATTGTGCTCCGATTATCGGTAAA GATGTTAGACAC SEQIDNo.87: GTCGAACATTGTGCTCCGATTATCGGTAAAGATGTTAGACACGTGTGCACGATTAATGAACCTAACATGATCG CCGTAATGGCGGGCTTAGCTAAGACAGGCGATCAAGGTTTCCCACCGGCGGGTTTGCCTACGCCTGACGAAGA AACCAC SEQIDNo.88: GGGTTTGCCTACGCCTGACGAAGAAACCACTCATGCTGTTATTGCTGCACATCACGCCGCGGTCAAAGCAGTA CGTGCCATTGATCCGGACATCCAGGTCGGCTGGACCATCGCTAATCAAGTATATCAGGCATTACCTGGTGCCG AAGATGTTACTG SEQIDNo.89: ATCAGGCATTACCTGGTGCCGAAGATGTTACTGCTGCATATCGTTACCCAAGAGAAGACGTGTTCATTGAAGC CGCTCGTGGCGATGACTGGATCGGCGTGCAATCTTACACACGCACGAAGATTGGTGCGGATGGCCCAATCCCG GCGCCTGAAGACGCTGAACGCACCCTGACTCAGTGGGAATATTACCC SEQIDNo.90: CTGAACGCACCCTGACTCAGTGGGAATATTACCCAGCTGCTGTTGGTCATGCTCTGCGTCACACAGCGGATGT CGCTGGCCCAGACATGCCGTTAATTGTAACCGAAAACGGTATCGCCACTGCGGATGACGCACGCCGTGTGGAT TATTACACTGGTGCACTGGAAGCCGTTTCAGCCGCGTTA SEQIDNo.91: GCACTGGAAGCCGTTTCAGCCGCGTTAGAAGATGGTGTGAATATTCATGGCTATCTGGCGTGGAGCGCTTTGG ATAACTATGAATGGGGTAGTTACAAACCGACTTTTGGCCTGATCGCAGTTGATCCTGTGACATTCGAAAGAAC GGCCAAG SEQIDNo.92: CAGTTGATCCTGTGACATTCGAAAGAACGGCCAAGCCGTCAGCAGTGTGGTTAGGTGAAATGGGTAGAACAAG ACAGTTGCCAAGAGCGGAACGCGGGAAGGGTGGGCGCGCCGACCCGGATCGACGAGAGCAGCGCGACTGG

(82) 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.

(83) 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.

(84) 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.