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Long PCR Methodology 173
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Long PCR Methodology
Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell
1. Introduction
In this chapter, we detail protocols of long polymerase chain reaction (PCR) and
long RT-PCR, which we have found to be versatile, sensitive, and straightforward to
optimize. We have used these protocols with success on several different templates,
including lambda phage DNA, HAV, HBV, HCV (1), torovirus (2), coxsackie B6
virus (3), and human beta galactosidase mRNA (R. Tellier, unpublished data). The
guanine–cytosine (GC) content of these templates varied from 37.8 to 58.8%. These
protocols have also been used on genomic human DNA with success (4).
The long PCR protocol we use is derived from the method described by Barnes
(5) for KLA-16, a mixture of KlenTaq 1 and Pfu. We have replaced this mix by a
commercially available mix, Advantage KlenTaq Polymerase mix (Clontech), a mix
of KlenTaq 1, and Deep Vent. In our hands, this mix was found to produce more
consistent results. KlenTaq 1 is an N-terminal deletion mutant of Taq, analogous to
the Klenow fragment enzyme, and devoid of the 5′- 3′ exonuclease activity of Taq (5).
Unexpectedly, KlenTaq 1 was also found to be more thermostable and slightly more
accurate than Taq (5,6), as well as less sensitive to variation in Mg 2+ concentration
and more processive than Taq (7).
2. Materials
1. Water free of RNase, DNAse, and proteinase (ddH 2 O). Aliquot and store at –20°C.
2. DTT (100 mM, Promega or Gibco BRL). Store at –20°C.
3. RNasin (20–40 U/µL; Promega). Store at –20°C.
4. Superscript II reverse transcriptase (Gibco BRL/Life Technologies). Store at –20°C.
5. 5× RT buffer (Gibco BRL). Store at –20°C.
6. RNase H (1–4 U/µL; Gibco BRL). Store at –20°C.
7. RNase T1 (900–3000 U/µL; Gibco BRL). Store at –20°C.
8. 50× Advantage KlenTaq Polymerase Mix (Clontech). Store at –20°C(see Note 1).
9. 10× KlenTaq PCR buffer (Clontech). Store at –20°C (see Note 2).
10. dNTP mix (10 mM each). Prepared in ddH 2 O from 100 mM dNTP set (Pharmacia). Aliquot
and store at –80°C.
11. Mineral oil (molecular biology grade, Sigma).
12. Primers. Aliquot at a concentration of 10 µM in ddH 2 O, store at –20°C (see Note 3).
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
173