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Nucleic Acids Research 2005 33(22):e188; doi:10.1093/nar/gni187
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Published online 9 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA

Petra Langerak, Anders O. H. Nygren1, Jan P. Schouten1 and Heinz Jacobs*

Division of Immunology, The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 1MRC-Holland bv 1057 SN Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 20 512 2066; Fax: +31 20 512 2057; Email: H.Jacobs{at}nki.nl

Received October 27, 2005. Revised November 21, 2005. Accepted November 21, 2005.

Embryonic stem (ES) cell technology allows modification of the mouse germline from large deletions and insertions to single nucleotide substitutions by homologous recombination. Identification of these rare events demands an accurate and fast detection method. Current methods for detection rely on Southern blotting and/or conventional PCR. Both the techniques have major drawbacks, Southern blotting is time-consuming and PCR can generate false positives. As an alternative, we here demonstrate a novel approach of Multiplex Ligation-dependent Probe Amplification (MLPA) as a quick, quantitative and reliable method for the detection of homologous, non-homologous and incomplete recombination events in ES cell clones. We have adapted MLPA to detect homologous recombinants in ES cell clones targeted with two different constructs: one introduces a single nucleotide change in the PCNA gene and the other allows for a conditional inactivation of the wild-type PCNA allele. By using MLPA probes consisting of three oligonucleotides we were able to simultaneously detect and quantify both wild-type and mutant alleles.

The author wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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