Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)
- 1Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA;
- 2Division of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110, USA;
- 3Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria
Abstract
Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.
Footnotes
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↵4 Corresponding author.
E-mail Kopan{at}wustl.edu; fax (314) 747-5503.
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[Supplemental material is available online at http://www.genome.org.]
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Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.093856.109.
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- Received March 16, 2009.
- Accepted July 30, 2009.
- Copyright © 2009 by Cold Spring Harbor Laboratory Press
