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dc.contributor.authorCastellanos-Rizaldos, Elena
dc.contributor.authorMilbury, Coren Audrey
dc.contributor.authorMakrigiorgos, G. Mike
dc.date.accessioned2013-04-24T20:28:09Z
dc.date.issued2012
dc.identifier.citationCastellanos-Rizaldos, Elena, Coren Audrey Milbury, and G. Mike Makrigiorgos. 2012. Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion. PLoS ONE 7(12): e51362.en_US
dc.identifier.issn1932-6203en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10582096
dc.description.abstractBackground: Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens. Methods: Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature \((T_c)\) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion. Results: Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined. Conclusions: TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.en_US
dc.language.isoen_USen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.isversionofdoi:10.1371/journal.pone.0051362en_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516544/pdf/en_US
dash.licenseLAA
dc.subjectBiologyen_US
dc.subjectBiochemistryen_US
dc.subjectNucleic Acidsen_US
dc.subjectDNAen_US
dc.subjectDNA amplificationen_US
dc.subjectBiophysicsen_US
dc.subjectGeneticsen_US
dc.subjectCancer Geneticsen_US
dc.subjectMolecular Cell Biologyen_US
dc.subjectPhysicsen_US
dc.subjectScience Policyen_US
dc.subjectTechnology Developmenten_US
dc.titleEnrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsionen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalPLoS ONEen_US
dash.depositing.authorMakrigiorgos, G. Mike
dc.date.available2013-04-24T20:28:09Z
dc.identifier.doi10.1371/journal.pone.0051362*
dash.contributor.affiliatedMakrigiorgos, Gerassimos


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