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Thermal Asymmetric PCR 355
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Direct Sequencing by Thermal Asymmetric PCR
Georges-Raoul Mazars and Charles Theillet
1. Introduction
Direct sequencing of polymerase chain reaction (PCR) products (1) has proven to
be a powerful method in the generation of nucleic acid sequence data. Using these
techniques, it is possible to produce microgram quantities of pure target DNA and
subsequently its nucleotide sequence in a few hours, even, theoretically, from one
single RNA or DNA molecule. However, problems have been encountered, and these
have been attributed to the strong tendency of the short double-strand DNA templates
to reanneal. In fact, compared with double-stranded plasmid DNA, which can be
permanently denatured by alkali treatment and then form intermolecular interactions
compatible with good sequencing efficiency, optimized conditions for direct sequencing
are required before reannealing with short PCR product.
To obviate this, strategies have been developed, such as the generation of singlestrand
DNA template by asymmetric PCR (2,3). Methods use either a disequilibrated
concentration ratio between the two primers or a two-step amplification, both of which
have their shortfalls. The first method is based on a large number of cycles, which is
a potential source of misincorporation of errors, and optimized conditions enough to
produce single-strand DNA that are strongly primer dependent. Moreover, it often has
been the case that only one strand can easily be sequenced. The second case requires
two physical separation steps, in which product contamination may occur.
Here, we propose a method combining the advantages of both symmetric and
asymmetric PCR. It is based on a thermal asymmetry between the Tm of both primers.
Annealing temperature of each primer is calculated with the formula: 69.3 + 0.41
(%GC) – 650/L, with L = primer length. PCR primers are designed to obtain a difference
in T m of at least 10°C. In the first step, double-stranded material is produced
during 20 to 25 cycles (to minimize the yield of spurious products) using the lower
Tm. During the second step, single-stranded DNA is generated using the higher
T m (Fig. 1).
Consequently, one primer is dropped out and linear amplification is obtained.
The final quantity of single-stranded product is comparable with the one produced
by Gyllensten and Erlich’s method (2). We applied thermal asymmetry to several
sequences, which, in our hands, were difficult to sequence both from double-stranded
DNA or with the Gyllensten and Erlich asymmetric PCR products (Fig. 2).
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
355