Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies

Journal of Visualized Experiments

Volumn , Issue 63, 2012, Pages

  Lorenz, Todd C ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

Additives; Basic protocols; Enhancers; Issue 63; Melting temperature; Optimization; PCR; Primer design; T m; Template; Troubleshooting

Indexed keywords

ARTICLE; METHODOLOGY; POLYMERASE CHAIN REACTION;

POLYMERASE CHAIN REACTION;

EID: 84863759926     PISSN: 1940087X     EISSN: None     Source Type: Journal    
DOI: 10.3791/3998     Document Type: Article

References (38)

1. Albert, J. & Fenyo, E.M. Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers. J. Clin. Microbiol. 28, 1560-1564 (1990).
2. Baskaran, N., et al. Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6, 633-638 (1996).
3. Blanchard, M.M., Taillon-Miller, P., Nowotny, P., & Nowotny, V. PCR buffer optimization with uniform temperature regimen to facilitate automation. PCR Methods Appl. 2, 234-240 (1993).
4. Borer, P.N., Dengler, B., Tinoco, I., Jr., & Uhlenbeck, O.C. Stability of ribonucleic acid double-stranded helices. J. Mol. Biol. 86, 843-853, [p2] 0022-2836(74)90357-X (1974).
5. Breslauer, K.J., Frank, R., Blocker, H., & Marky, L.A. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750 (1986).
6. Chou, Q., Russell, M., Birch, D.E., Raymond, J., & Bloch, W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copynumber amplifications. Nucleic Acids Res. 20, 1717-1723 (1992).
7. Coleman, W.B.A.T. & Gregory, J. 592, Humana Press, (2005).
8. D'Aquila, R.T., et al. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Res. 19, 3749 (1991).
9. Dieffenbach, C.W., Lowe, T.M., & Dveksler, G.S. General concepts for PCR primer design. PCR Methods Appl. 3, S30-37 (1993).
10. Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., & Mattick, J.S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 (1991).
11. Erlich, H.A., Gelfand, D., & Sninsky, J.J. Recent advances in the polymerase chain reaction. Science. 252, 1643-1651 (1991).
12. Frey, U.H., Bachmann, H.S., Peters, J., & Siffert, W. PCR-amplification of GC-rich regions: 'slowdown PCR'. Nat. Protoc. 3, 1312-1317, doi:10.1038/nprot.2008.112 [pii] nprot.2008.112 (2008).
13. Haqqi, T.M., Sarkar, G., David, C.S., & Sommer, S.S. Specific amplification with PCR of a refractory segment of genomic DNA. Nucleic Acids Res. 16, 11844 (1988).
14. Henke, W., Herdel, K., Jung, K., Schnorr, D., & Loening, S.A. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957-3958, [p2] gka606 (1997).
15. Innis, M.A., Gelfand, David H., Sninsky, John J., & White, Thomas J. 482 Academic Press, Inc., San Diego, (1990).
16. King, N. In: Methods in Molecular Biology. Vol. 630, Humana Press, (2010).
17. Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I., & Khorana, H.G. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases. J. Mol. Biol. 56, 341-361 (1971).
18. Korbie, D.J. & Mattick, J.S. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat. Protoc. 3, 1452-1456, doi:10.1038/nprot.2008.133 [pii] nprot.2008.133 (2008).
19. Kramer, M.F. & Coen, D.M. Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr. Protoc. Toxicol. Appendix 3, A 3 C 1-14, doi:10.1002/0471140856.txa03cs03 (2001).
20. Kramer, M.F. & Coen, D.M. The polymerase chain reaction. Curr. Protoc. Protein Sci. Appendix 4, Appendix 4J, doi:10.1002/0471140864.psa04js29 (2002).
21. Kreader, C.A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102-1106 (1996).
22. Kwok, S., et al. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J. Virol. 61, 1690-1694 (1987).
23. McConlogue, L., Brow, M.A., & Innis, M.A. Structure-independent DNA amplification by PCR using 7-deaza-2′-deoxyguanosine. Nucleic Acids Res. 16, 9869 (1988).
24. Mullis, K.B. The unusual origin of the polymerase chain reaction. Sci. Am. 262, 56/61, 64-55 (1990).
25. Mullis, K.B. Target amplification for DNA analysis by the polymerase chain reaction. Ann. Biol. Clin. (Paris). 48, 579-582 (1990).
26. Mullis, K.B. The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion. PCR Methods Appl. 1, 1-4 (1991).
27. Mullis, K B. & Faloona, F.A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-350 (1987).
28. Paabo, S., Gifford, J.A., & Wilson, A.C. Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res. 16, 9775-9787 (1988).
29. Rees, W.A., Yager, T.D., Korte, J., & von Hippel, P. H. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 32, 137-144 (1993).
30. Roux, K.H. & Hecker, K.H. One-step optimization using touchdown and stepdown PCR. Methods Mol. Biol. 67, 39-45 (1997).
31. Rychlik, W., Spencer, W.J., & Rhoads, R.E. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409-6412 (1990).
32. Saiki, R.K., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 239, 487-491 (1988).
33. Saiki, R.K., et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 230, 1350-1354 (1985).
34. Sambrook, J.A.R., & David, W. Molecular Cloning A Laboratory Manual. 3rd edn., Vol. 2, Cold Spring Harbor Laboratory Press (2001).
35. Sarkar, G., Kapelner, S., & Sommer, S.S. Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. 18, 7465 (1990).
36. Smit, V.T., et al. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res. 16, 7773-7782 (1988).
37. Wilson, I.G. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741-3751 (1997).
38. Wu, R. In: Methods in Enzymology. Vol. 218 806, Academic Press, Inc, San Diego, (1993).