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Subcycling PCR 101
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Subcycling PCR for Long-Distance Amplifications
of Regions with High and Low Guanine–Cystine Content
Amplification of the Intron 22 Inversion of the FVIII Gene
David Stirling
1. Introduction
Hemophilia A is an X-linked disorder caused by mutations in the factor VIII gene.
Around 50% of all patients with severe hemophilia A share a common mutation. This
intron 22 inversion results from homologous recombination of a sequence within
intron 22 of the factor VIII gene and identical sequence around 500 kb telomeric to
the gene. Although this inversion could be detected by Southern blotting, the development
of a long polymerase chain reaction (PCR) assay, and its subsequent improvement
by subcycling PCR (S-PCR), greatly facilitated the provision of diagnostic
services (1,2).
In S-PCR, the annealing/elongation step is composed of subcycles of shuttling
between a low and a high temperature. S-PCR produces consistent amplification of the
various segments produced by wild-type, mutant, and carrier individuals. S-PCR is a
robust variant of PCR, which may be of use in amplification of long segments in which
the guanine–cytosine (GC) content varies among the segments, multiplex amplification
of long segments, and multiplex amplification of short segments in which the GC
content varies among the segments. We have also found it greatly improves the success
of amplification from partially degraded templates.
2. Materials
Except where otherwise stated, all reagents are from Sigma Chemical Company,
Poole, UK.
1. Thermal cycler.
2. Expand Long Template PCR System (Roche).
3. Dimethyl sulfoxide.
4. DNTP (Promega) Supplied separately (dATP, dCTP, dGTP, dTTP) at concentrations of
100 mM. Use at 10 mM. Aliquot 10 µL of stock dNTP into labeled tubes containing 90 µL
of sterile distilled water. Store at –20°C.
5. 7-Deaza GTP (Roche).
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
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