Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA.
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.
Langerak
Petra
P
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Nygren
Anders O H
AO
Schouten
Jan P
JP
Jacobs
Heinz
H
D000818:Animals; D015139:Blotting, Southern; D002999:Clone Cells; D004247:DNA / Q000032:analysis; D004622:Embryo, Mammalian / Q000166:cytology; D018390:Gene Targeting / Q000379:methods*; D051379:Mice / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D011995:Recombination, Genetic; D013234:Stem Cells / Q000737:chemistry
D015345:Oligonucleotide Probes; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA