Amplification Ratio Control System for Copy Number Variation Genotyping

Philip A I Guthrie, TR Gaunt, MR Abdollah, S Rodriguez, D Lawlor, G Davey Smith, INM Day

Research output: Contribution to journalArticle (Academic Journal)peer-review

8 Citations (Scopus)

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 (UP0) in each strand form a hairpin. The bases immediately beyond the 30-end of the UP and 50 of UP0 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 30-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.
Translated title of the contributionAmplification Ratio Control System (ARCS) for Copy Number Variation Genotyping
Original languageEnglish
Article numbere54
Number of pages12
JournalNucleic Acids Research
Volume39
Issue number8
DOIs
Publication statusPublished - 7 Feb 2011

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