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Nucleic Acids Res. 2011 Apr;39(8):e54. doi: 10.1093/nar/gkr046. Epub 2011 Feb 7.

Amplification ratio control system for copy number variation genotyping.

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1
Bristol Genetic Epidemiology Laboratory and MRC Centre for Causal Analyses in Translational Epidemiology, School of Social and Community Medicine, University of Bristol, Oakfield Grove, Clifton BS8 2BN, UK. philip.guthrie@bristol.ac.uk

Abstract

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.

PMID:
21300641
PMCID:
PMC3082892
DOI:
10.1093/nar/gkr046
[Indexed for MEDLINE]
Free PMC Article
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