Nucleic Acids Research Advance Access originally published online on December 14, 2006
Nucleic Acids Research 2007 35(3):e19; doi:10.1093/nar/gkl1089
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Nucleic Acids Research, 2007, Vol. 35, No. 3 e19
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats
Institute of Genetics, University of Nottingham Nottingham, NG7 2UH, UK 1 Department of Dermatology, Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands 2 Department of Endocrinology and Department of Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands 3 Department of Genetics, University of Leicester University Road, Leicester LE1 7RH, UK
*To whom correspondence should be addressed. Tel: +44 115 8230308; Fax: +44 115 8230313; Email: john.armour{at}nottingham.ac.uk
Received October 7, 2006. Revised November 28, 2006. Accepted November 28, 2006.
Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.
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