Circular Polymerase Extension Cloning for High-throughput Cloning of Complex and Combinatorial DNA Libraries

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2011 Feb 5

PMID 21293463

Citations 154

Authors

Jiayuan Quan

Jingdong Tian

Affiliations

Soon will be listed here.

Abstract

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.

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References

Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X . Accurate multiplex gene synthesis from programmable DNA microchips. Nature. 2004; 432(7020):1050-4. DOI: 10.1038/nature03151. View

Gibson D, Benders G, Axelrod K, Zaveri J, Algire M, Moodie M . One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci U S A. 2008; 105(51):20404-9. PMC: 2600582. DOI: 10.1073/pnas.0811011106. View

Breslauer K, Frank R, Blocker H, Marky L . Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986; 83(11):3746-50. PMC: 323600. DOI: 10.1073/pnas.83.11.3746. View

Cohen S, Chang A, Boyer H, Helling R . Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973; 70(11):3240-4. PMC: 427208. DOI: 10.1073/pnas.70.11.3240. View

Aslanidis C, de Jong P . Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 1990; 18(20):6069-74. PMC: 332407. DOI: 10.1093/nar/18.20.6069. View

Quan J, Tian J . Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 2009; 4(7):e6441. PMC: 2713398. DOI: 10.1371/journal.pone.0006441. View

Rashtchian A . Novel methods for cloning and engineering genes using the polymerase chain reaction. Curr Opin Biotechnol. 1995; 6(1):30-6. DOI: 10.1016/0958-1669(95)80006-9. View

Smith H, Hutchison 3rd C, Pfannkoch C, Venter J . Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A. 2003; 100(26):15440-5. PMC: 307586. DOI: 10.1073/pnas.2237126100. View

Stemmer W, Crameri A, Ha K, Brennan T, Heyneker H . Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995; 164(1):49-53. DOI: 10.1016/0378-1119(95)00511-4. View

10.

Garces C, Laborda J . Single-step, ligase-free cloning of polymerase chain reaction products into any restriction site of any DNA plasmid. Anal Biochem. 1995; 230(1):178-80. DOI: 10.1006/abio.1995.1454. View

11.

Horton R, Hunt H, Ho S, Pullen J, Pease L . Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989; 77(1):61-8. DOI: 10.1016/0378-1119(89)90359-4. View

12.

Li M, Elledge S . Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods. 2007; 4(3):251-6. DOI: 10.1038/nmeth1010. View

13.

Walhout A, Temple G, Brasch M, HARTLEY J, Lorson M, Van den Heuvel S . GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 2000; 328:575-92. DOI: 10.1016/s0076-6879(00)28419-x. View

14.

Shevchuk N, Bryksin A, Nusinovich Y, Cabello F, Sutherland M, Ladisch S . Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 2004; 32(2):e19. PMC: 373371. DOI: 10.1093/nar/gnh014. View

15.

Prodromou C, Pearl L . Recursive PCR: a novel technique for total gene synthesis. Protein Eng. 1992; 5(8):827-9. DOI: 10.1093/protein/5.8.827. View

16.

McArthur 4th G, Fong S . Toward engineering synthetic microbial metabolism. J Biomed Biotechnol. 2009; 2010:459760. PMC: 2796345. DOI: 10.1155/2010/459760. View

17.

Shao Z, Zhao H, Zhao H . DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 2008; 37(2):e16. PMC: 2632897. DOI: 10.1093/nar/gkn991. View

18.

Nisson P, Rashtchian A, Watkins P . Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Methods Appl. 1991; 1(2):120-3. DOI: 10.1101/gr.1.2.120. View

19.

Scharf S, Horn G, Erlich H . Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986; 233(4768):1076-8. DOI: 10.1126/science.3461561. View

20.

HARTLEY J, Temple G, Brasch M . DNA cloning using in vitro site-specific recombination. Genome Res. 2000; 10(11):1788-95. PMC: 310948. DOI: 10.1101/gr.143000. View