Preferential amplification is minimised in long-PCR systems.
The advent of long PCR (XL-PCR) has proven to be a major advance in PCR technology and is currently being utilised to investigate numerous biological systems. The analysis of mixed DNA populations is a particularly useful application for XL-PCR. For example, XL-PCR has been used to investigate the occurrence of heterogeneous mitochondrial DNA (mtDNA) rearrangement mutations. With XL-PCR it became possible to amplify the entire length of the mtDNA chromosome and detect any mtDNA deletion or insertion mutations based on a measurable change in overall sequence length. In the present communication, XL-PCR and conventional short-length PCR were used to amplify mitochondrial DNA sequences from several human vastus lateralis skeletal muscle samples. The experiments demonstrated that there was minimal preferential amplification of shorter DNA sequences with XL-PCR and was significantly less than the preferential amplification of shorter sequences observed with conventional PCR. Also, XL-PCR amplification of the complete mtDNA sequence from control DNA containing a single mtDNA template (leucocyte extracts) showed that the generation of PCR artefacts was not a predisposed failing of the system but was dependant on the standard rules that govern the set up and optimisation of any PCR reaction. In optimised systems, XL-PCR artefacts were not generated and a single PCR product was always recovered.
Kopsidas
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Centre for Molecular Biology and Medicine, Epworth Medical Centre, 185-187 Hoddle Street, Richmond, Vic. 3121, Australia. gkopsidas@cmbm.com.au
Kovalenko
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Islam
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Gingold
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Linnane
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D000375:Aging / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D005784:Gene Amplification; D015321:Gene Rearrangement; D006801:Humans; D018482:Muscle, Skeletal / Q000737:chemistry; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004272:DNA, Mitochondrial