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Long PCR 167
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Long PCR Amplification of Large Fragments
of Viral Genomes
A Technical Overview
Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell
1. Introduction
1.1. Long PCR
The polymerase chain reaction (PCR) has become an essential and ubiquitous
tool for biological research and laboratory diagnostic applications. Until recently,
reliable and sensitive amplification of large templates (several kb) was difficult to
achieve. However, in 1994, an important breakthrough was reported by Barnes (1).
He hypothesized that a major obstacle to long PCR was the Taq DNA polymerase
error rate, which causes mismatches that make elongation very inefficient. Many other
thermostable DNA polymerases have a 3′ to 5′ exonuclease “proofreading” activity and
a higher fidelity. However, the use of these polymerases alone does not reliably achieve
long PCR, presumably because of excessive degradation of primers by the exonuclease
activity (1). The processivity of the enzyme may also be a factor. Of note, the 3′ to 5′
exonuclease activity alone is not a guarantee of high fidelity: Fidelity also depends
on the degree of discrimination against misinsertion, the mismatch extension rate,
and the rate of shuttling between polymerizing and proofreading modes (2). The
breakthrough reported by Barnes consisted in performing PCR with a mixture of
two DNA polymerases: a major component consisting of a highly processive DNA
polymerase and a minor component consisting of a DNA polymerase with a 3′ to 5′
exonuclease “proofreading” activity. With such enzyme mixes, reliable amplification
of templates up to 35 kb in length was achieved (1). The greater fidelity of long PCR
enzyme mixes, relative to Taq, has been demonstrated (1,2). Other modifications
contribute to making long PCR possible, including optimization of the buffer and the
thermal cycling conditions. It is especially important to address the drop in pH at high
temperature observed with Tris-based buffers (1,3). This is significant because DNA
depurination is enhanced both by high temperature and by low pH. Because a larger
DNA template is more likely than a small template to sustain depurination at sites
within its boundaries, long PCR is inherently more vulnerable to depurination (1,3).
Interestingly, in contrast to 3′ to 5′ exonuclease-deficient DNA polymerases, the fidelity
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
167