Amplification ratio control system for copy number variation genotyping
Amplification ratio control system for copy number variation genotyping
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
e54
Guthrie, Philip A.I.
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Gaunt, Tom R.
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Abdollahi, Mohammed R.
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Rodriguez, Santiago
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Lawlor, Debbie A.
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Smith, George Davey
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Day, Ian N.M.
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April 2011
Guthrie, Philip A.I.
2ad09527-826e-4d74-bb50-0962dacb0292
Gaunt, Tom R.
ff4bc39d-405c-4ba1-896b-7e7d2f747387
Abdollahi, Mohammed R.
96ec68db-6302-4216-9aaf-9951a44be8b8
Rodriguez, Santiago
f235ea2b-b6f3-45e4-9fc3-5a0383689ed6
Lawlor, Debbie A.
799826df-f115-4fb7-83ea-53c246c220d4
Smith, George Davey
f5bc8327-f2cb-49a0-8eae-4a6ba63207a2
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Guthrie, Philip A.I., Gaunt, Tom R., Abdollahi, Mohammed R., Rodriguez, Santiago, Lawlor, Debbie A., Smith, George Davey and Day, Ian N.M.
(2011)
Amplification ratio control system for copy number variation genotyping.
Nucleic Acids Research, 39 (8), .
(doi:10.1093/nar/gkr046).
(PMID:21300641)
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
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Published date: April 2011
Organisations:
Cancer Sciences
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Local EPrints ID: 364361
URI: http://eprints.soton.ac.uk/id/eprint/364361
ISSN: 0305-1048
PURE UUID: 3936dfa8-da9f-49a5-9374-1ab254491a01
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Date deposited: 25 Apr 2014 08:54
Last modified: 14 Mar 2024 16:34
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Contributors
Author:
Philip A.I. Guthrie
Author:
Tom R. Gaunt
Author:
Mohammed R. Abdollahi
Author:
Santiago Rodriguez
Author:
Debbie A. Lawlor
Author:
George Davey Smith
Author:
Ian N.M. Day
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