COLD-PCR: improving the sensitivity of molecular diagnostics assays

Milbury, Coren A.; Li, Jin; Liu, Pingfang; Makrigiorgos, G. Mike

doi:10.1586/erm.10.115

Cited by 51 publications

(35 citation statements)

References 43 publications

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“…190-plex fast-TT-COLD- PCR and conventional PCR were compared by examining a wild type sample and mutant serial dilutions (5%, 2.5%, 1.25%, 0.63%, 0.31% and 0.16%): a gene-specific PCR was performed after conventional PCR and fast-TT-COLD- PCR for exon 5 of TP53 , followed by HRM. Conventional PCR followed by HRM had a limit of detection of 2.5% mutation abundance, consistent with previous reports (36). Fast-TT-COLD-PCR on the other hand discriminated differences from wild type DNA down to 0.31% mutation abundance (Supplemental Figure 4).…”

Section: Resultssupporting

confidence: 91%

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

Castellanos-Rizaldos¹,

Richardson

Lin

et al. 2015

Clinical Chemistry

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BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-re-sequencing. Analysis of mutations below ~2–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids, is problematic due to increased ‘noise’ from sequencing errors. Technologies that reduce noise via deep-sequencing unavoidably reduce throughput and increase cost. Here we provide proof-of-principle that COLD-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS We have developed a multiplex temperature-tolerant-COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. Following a multiplex pre-amplification from genomic DNA we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for Tm-increasing as well as Tm-decreasing mutations over all amplicons, in a single tube. RESULTS Using custom-made and commercial gene panels containing 8, 50, 190 or 16,000 amplicons we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of dITP/dDTP in place of dGTP/dATP enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit-of-detection of ~ 0.01–0.1% mutation abundance using Ion-Torrent and 0.1–0.3% using Sanger sequencing. CONCLUSIONS Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

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Section: Resultssupporting

confidence: 91%

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

Castellanos-Rizaldos¹,

Richardson

Lin

et al. 2015

Clinical Chemistry

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“…3 In contrast, when there is no minor allele (for example a heterozygous SNP), there will be no change in allele following COLD-PCR. Finally, our method depends on the fact that HRM data can be used to detect the changes in minor allele frequency.…”

Section: 713-15mentioning

confidence: 99%

“…Finally, our method depends on the fact that HRM data can be used to detect the changes in minor allele frequency. 3,[20][21][22][23][24] HRM is a simple and robust method for detection of sequence mutations such as point mutations, small indels (including microsatellite instability), and gene promoter methylation (following bisulphite treatment). 17,18,[25][26][27][28][29] The underlying principle is that mutant and wild-type DNA sequences can be pushed into forming heteroduplexes which melt differently to homoduplexes.…”

Section: 713-15mentioning

confidence: 99%

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COLD-HRM: A Combination of Methods to Infer the Nature of Somatic Mutations

Ham-Karim

Ebili

Fadhil

et al. 2017

ACP

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Abbreviations: COLD PCR, CO-amplification at lower denaturation temperature PCR; LOH, loss of heterozygosity; HRM, high resolution melting; SSCP, single strand conformation analysis IntroductionLoss of Heterozygosity (LOH) and sequence mutation are two frequently occurring types of somatic alterations in cancer. 1-3Detection of these changes is important as it delineates the profile of a cancer and it may also provide prognostic and predictive information.4-12 A variety of techniques exist for detection of these mutations. LOH can be detected, at the gross chromosomal level, with techniques such as array-based chromosomal genomic hybridization (aCGH), karyotyping and fluorescent in-situ hybridization (FISH). 1,7-9For a higher resolution analysis, genotyping through quantification of Single Nucleotide Polymorphisms (SNPs) or microsatellites can be performed.13-15 These methods of detecting LOH can be cumbersome and often require matching non-tumour DNA from the same patient. 1,7,13-15The gold standard for detecting somatic sequence mutations is DNA sequencing.16 However, unless there is a well-defined and narrow mutation hot-spot (such as mutations of codon 12/13 in KRAS), large areas of DNA may need to the tested costing both time and money. Costs can be reduced by screening the DNA for the possible presence of mutations using techniques such as High Resolution Melting (HRM), Single Strand Conformation Analysis (SSCP) and denaturing High Performance Liquid Chromatography (dHPLC).17-19 Such methods interrogate the physical characteristics of the DNA which are altered by a change in the DNA sequence. These methods cannot however distinguish between germline SNPs and cancer-specific somatic changes unless normal DNA from the patient is available for comparison.The currently available techniques for detecting LOH and somatic sequence mutations can be time-consuming and expensive and an improved methodology-in particular one which would remove the requirements for samples of matched normal DNA-is required. In this paper, we report developments to achieve this. Our method depends firstly on the fact that both LOH and sequence mutation induce a state of major and minor alleles of the target gene 1-3 in a tumour. LOH forces a minority state on the wild type allele due to loss of chromatin or gene conversion whilst, in the case of somatic sequence mutation, the presence of contaminating wild type DNA from stromal cells, will result in the mutant allele becoming a minor allele.1-3 Secondly, we used the fact that the frequency of the minor allele can change due to the minor allele-enrichment capability of the Co-Amplification at Lower Denaturation temperature (COLD) PCR protocol.3 In contrast, when there is no minor allele (for example a heterozygous SNP), there will be no change in allele following COLD-PCR. Finally, our method depends on the fact that HRM data can be used to detect the changes in minor allele frequency. 3,20-24HRM is a simple and robust method for detection of sequence mutations such as point mutations, ...

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“…Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wildtype and mutation-containing sequences, provides a general platform to improve the sensitivity of DNA variation detection technologies (5,6). DNA genotyping with mutation-specific TaqMan ® probes (Applied Biosystems) is broadly used in the detection of single-nucleotide polymorphisms but is less frequently used for somatic mutations due to its limited selectivity for low-level mutations (7).…”

Section: Introductionmentioning

confidence: 99%

Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan® -MGB probe genotyping method

Liu

Liang

Xue

et al. 2012

Experimental and Therapeutic Medicine

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Despite the improved ability to detect mutations in recent years, tissue specimens cannot always be procured in a clinical setting, particularly from patients with recurrence of tumors or metastasis. Therefore, the aim of this study was to investigate whether plasma is able to be used for mutation analysis instead of tissue specimens. We collected plasma from 62 patients with colorectal cancer (CRC) prior to treatment. DNA extracted from plasma and matched tumor tissues were obtained. Mutations in KRAS were amplified from the tissue specimens and sequenced by regular polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR. Plasma KRAS gene mutation on codon 12 (GGT>GAT) was detected using a nested COLD-PCR/TaqMan(®) -MGB probe. Mutations in plasma and matched tumors were compared. KRAS mutation on codon 12 (GGT>GAT) was found in 13 (21.0%) plasma specimens and 12 (19.4%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors was 75% (9/12), which indicated a high correlation between the mutations detected in plasma DNA and the mutations detected in the corresponding tumor DNA (P<0.001; correlation index, k=0.649). Notably, four (6.5%) patients with plasma DNA mutations had no detectable KRAS mutations in the corresponding primary tumors, and three (4.8%) patients with tumor DNA mutations had no detectable KRAS mutations in the corresponding plasma DNA samples. Thus, KRAS mutations in plasma DNA correlate with the mutation status in matched tumor tissues of patients with CRC. Our study provides evidence to suggest that plasma DNA may be used as a potential medium for KRAS mutation analysis in CRC using the COLD-PCR/TaqMan-MGB probe method.

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COLD-PCR: improving the sensitivity of molecular diagnostics assays

Cited by 51 publications

References 43 publications

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

COLD-HRM: A Combination of Methods to Infer the Nature of Somatic Mutations

Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan® -MGB probe genotyping method

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