Abstract
BackgroundMultiplex 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.MethodsFollowing 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 (Tc) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion.ResultsValidation 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.ConclusionsTT-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.
Highlights
Detection of low-level mutant alleles in the presence of wild-type DNA is important in several fields of medicine, including cancer, pre-natal diagnosis and infectious diseases [1]
A main advantage of this procedure is its simplicity, since it does not require new instrumentation, special reagents or additional processing. This makes COLD-PCR accessible to different laboratory settings [3] that employ a variety of downstream mutation detection assays including high resolution melting, HRM [4,5,6,7,8], Sanger sequencing [9,10,11,12], mass-spectrometry [2], real time PCR [13,14] and RFLP analysis [15]
In this work we demonstrate that the problem of multiplexing COLD-PCR can be overcome by employing temperature-tolerant amplification in emulsion
Summary
Detection of low-level mutant alleles in the presence of wild-type DNA is important in several fields of medicine, including cancer, pre-natal diagnosis and infectious diseases [1]. A number of approaches have been described that enrich the abundance of lowlevel mutant allelic burden present in biological specimens, so that mutations can subsequently be detected [1] These include electrophoretic or enzymatic approaches, which require multiple reagents or specialized equipment. 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. 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; it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens
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