Long‐range PCR

Abstract

The polymerase chain reaction (PCR) has been used extensively in the amplification of DNA fragments where there is a general size preference for products less than 1000 bp. Larger products may be synthesized at low or inefficient levels and then normally after considerable effort in reaction optimization. While Taq DNA polymerase has remained the thermostable polymerase of choice for most PCRs, this enzyme is not efficient in the amplification of large DNA fragments. This limitation is thought to be due to the error‐prone nature of the Taq polymerase, whereby those transcripts that end in an error (a misincorporated base) are not efficiently reinitiated and extended. The combination of two thermostable DNA polymerases such as Taq and another enzyme with superior editing (proof‐reading) ability minimizes misincorporation and facilitates the amplification of long (reportedly over 40 kb) PCR products.

Keywords: DNA amplification; thermostable polymerase cocktail; Taq polymerase induced errors; proof‐reading

Figure 1.

Principle of long‐range PCR using thermostable DNA polymerases with proof‐reading and editing properties.

Figure 2.

RT‐PCR products from the neurofibromatosis 1 (NF1) gene fractionated on a 2% agarose gel. Lanes: M, Lambda DNA cleaved with Pst I; 1, 2 and 3, XL PCR products from cDNA prepared from NF1 patients; 4, 5 and 6, conventional PCR from the same cDNA preparations; 7, 8 and 9, conventional PCR of shorter NF1 transcripts from the same cDNA preparations.

Figure 3.

Addition of enzyme mixture to the long‐range PCR.

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References

Barnes WM (1994) PCR amplification of up to 35‐kb DNA with high fidelity and high yield from l bacteriophage templates. Proceedings of the National Academy of Sciences of the USA 91: 2216–2220.

Cheng S, Fockler C, Barnes WM, Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proceedings of the National Academy of Sciences of the USA 91: 5695–5699.

Eckert KA, Kunkel TA (1991) The fidelity of DNA polymerase and polymerases used in the PCR. In: McPherson MJ, Quirke P, Taylor GR (eds) Polymerase Chain Reaction I: A Practical Approach. Oxford: IRL Press.

Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction‐amplified DNA. Proceedings of the National Academy of Sciences of the USA 85: 9436–9440.

Further Reading

Saiki R, Gelfand DH, Stoeffel S et al. (1988) Primer‐directed enzymic amplification of DNA with thermostable polymerase. Science 239: 487–491.

Wilton SD, Lim L, Dorosz SM et al. (1995) Assignment of the human alpha tropomyosin gene (TPM4) to band 19p13.1 by fluorescent in situ hybridization. Cytogenetics and Cell Genetics 72: 294–296.

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How to Cite close
Wilton, S(Oct 2002) Long‐range PCR. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003767]