Subcycling-PCR for Multiplex Long-Distance Amplification of Regions with High and Low GC Content: Application to the Inversion Hotspot in the Factor VIII Gene

Abstract

Previously we described a PCR protocol for detecting the inversion in the factor VIII gene, which is a common cause of Hemophilia A. This PCR assay is challenging due to the size of the amplification (10–12 kb), the varying GC content (30%–80%) and the multiplex PCR products involved (four for carrier female). Efficient amplification of the four segments depends on three unusual modifications to standard long-distance PCR protocols: (i)very high concentrations of dimethyl sulfoxide, (ii)addition of deazadGTP and (iii) high concentration of Taq and PwoDNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 s at 94°C, 30 s at 65°C and 14 min at 68°C). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step is composed of subcycles of shuttling between a low and a high temperature, e.g., shuttling four times between 60° and 65°C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant and carrier individuals. S-PCR is a simple generalization of PCR, which generally may be advantageous in three contexts: (i) amplification of long segments in which the GC content varies within the segment, (ii) multiplex amplification of long segments and (iii) multiplex amplification of short segments in which the GC content varies among the segments.