Dataset Information

Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion.

ABSTRACT:

Background

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.

SUBMITTER: Castellanos-Rizaldos E

PROVIDER: S-EPMC3516544 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion.

Castellanos-Rizaldos Elena E Milbury Coren Audrey CA Makrigiorgos G Mike GM

PloS one 20121206 12

<h4>Background</h4>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 m ...[more]

PMID: 23236486

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Project description:Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.

Project description:BACKGROUND:Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. METHODS:We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. RESULTS:A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. CONCLUSIONS:Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

Dataset Information

Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion.

Background

Methods

Results

Conclusions

Publications

Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion.

Similar Datasets

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