Lower Denaturation Temperature PCR Research Articles

The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

A sensitive and convenient method for the detection of epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs) would be desirable to guide treatment strategy. Consequently, studies have focused on sensitive characterization of EGFR T790M mutation. Herein, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and pyrosequencing were combined (COLDPCR/ pyrosequencing) for detecting EGFR T790M mutation. Evaluation of mutation-containing dilutions revealed that the sensitivities of COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing assays for the detection of the T790M mutation were 0.1 and 5%, respectively, indicating a 50-fold increase in sensitivity. When the T790M mutation in 20 clinical NSCLC samples who had relapsed under firstgeneration EGFR TKI were further determined using COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing, the detection rates were 35% (7/20) and 25% (5/20), respectively. All patients who were positive for the T790M mutation with conventional PCR/pyrosequencing were also found to be positive with COLD-PCR/pyrosequencing. The discordant cases were 2 samples with no T790M mutation detected with conventional PCR/pyrosequencing, but which were positive with COLD-PCR/pyrosequencing. COLD-PCR/pyrosequencing is a sensitive and cost-effective tool for detecting the T790M mutation which will permit an improvement of therapeutic management.

Establishment of Coamplification at Lower Denaturation Temperature PCR/Fluorescence Melting Curve Analysis for Quantitative Detection of Hepatitis B Virus DNA, Genotype, and Reverse Transcriptase Mutation and Its Application in Diagnosis of Chronic Hepatitis B

Dynamic and real-time hepatitis B virus (HBV) DNA, genotype, and reverse transcriptase mutation analysis plays an important role in diagnosing and monitoring chronic hepatitis B (CHB) and in assessing the therapeutic response. We established a highly sensitive coamplification at lower denaturation temperature PCR (COLD-PCR) coupled with probe-based fluorescence melting curve analysis (FMCA) for precision diagnosis of CHB patients. The imprecision with %CV and detection limit of HBV DNA detected by COLD-PCR/FMCA were 2.58% to 4.42% and 500 IU/mL, respectively. For mutation, the imprecision and detection limit were 3.35% to 6.49% and 1%, respectively. Compared with Sanger sequencing, the coincidence rates of genotype and mutation were 96.0% and 82.5%, respectively, whereas the inconsistent data resulted from a low proportion (<20%) of mixed genotypes or mixed mutations. The mutation ratio in HBV infection patients was as follows: hepatitis B e antigen (HBeAg)-positive infection (0/0.0%)<HBeAg-negative infection (16/4.5%)<HBeAg-positive hepatitis (30/5.5%)<HBeAg-negative hepatitis (36/6.5%). In patients with entecavir therapy, the proportion of mutation at baseline or week 4 in virologic response (VR) group was <4%, whereas in the partial VR group, it was mostly≥4%. COLD-PCR/FMCA provides a novel tool with high sensitivity, convenience, and practicability for the simultaneous quantification of HBV DNA, genotype, and mutation. It might be used for distinguishing the different phases of HBV infection and predicting VR of CHB patients.

Sensitive molecular testing methods can demonstrate NSCLC driver mutations in malignant pleural effusion despite non-malignant cytology.

Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (KRAS) mutation demonstrated the same KRAS mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known KRAS mutation, ICECOLD PCR detected the same KRAS variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies.

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR)for the sensitive detection of disease-related hypermethylation.

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Co-amplification at lower denaturation temperature-PCR: methodology and applications.

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

Abstract 1390: High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR

Abstract Introduction: The EGFR Exon 20 T790M mutation confers resistance to the first generation tyrosine kinase inhibitors (TKI), such as Tarceva®. A new generation of treatments, including AZD9291, targets the T790M mutation thus allowing a new approach to cancer therapy for NSCLC patients. Recently EGFR Exon 20 C797S mutations (c.2389T&amp;gt;A and c.2390G&amp;gt;C) have been identified as resistance mutations to the third generation TKI AZD9291. High sensitivity detection of these mutations, as well as the T790M mutation, in patients with acquired resistance to third generation TKI is an important requirement for best practice NSCLC treatment. Materials and Methods: ICE COLD-PCR (Improved &amp; Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide (RS-oligo) complementary to the wild-type sequence. Due to the inherent sequence characteristics of the region between the codons of interest (790 and 797), a RS-oligo focusing on the C797S was developed and is independent of the RS-oligo for T790M, and thus tiling across EGFR Exon 20 can be performed. The RS-oligo in this assay was designed to prevent PCR amplification of wild-type sequences while allowing exponential amplification of any mutations including C797S within the RS-oligo binding region. Serial dilutions of spiked gBlocks® DNA containing C797S mutations mixed with K562 DNA and samples extracted from plasma were amplified with ICE COLD-PCR followed by Sanger sequencing analysis. Result: For mutations c.2389T&amp;gt;A and c.2390G&amp;gt;C, limits of detection as low as 0.05% were achieved. Forty DNA samples extracted from plasma were also tested. These indicated that the assay is suitable for fragmented samples with low quantities of input DNA such as liquid biopsies. Conclusion: The pre-amplification PCR product covers both codons 790 and 797. This allows ICE COLD-PCR to be used for the analysis of both T790M and C797S using the same liquid biopsy sample aliquot. This new ICE COLD-PCR method to detect both C797S mutations c.2389T&amp;gt;A and c.2390G&amp;gt;C may provide a highly sensitive, simple and cost effective assay for monitoring acquired resistance to AZD9291 in the liquid biopsies obtained from patients and clinical trials. Coupled with our existing highly sensitive assay for T790M, ICE COLD-PCR offers a highly sensitive method to determine the emergence of resistance mutations, thus potentially contributing to therapeutic optimization. Citation Format: Grant Wu, Sheena Jensen, Karissa Scott, Erin Montagne, Jason Stoddard, Phil Krzycki, Courtney Schweikart, Benjamin L. Legendre, Philip Eastlake, Katherine A. Richardson. High sensitivity detection of EGFR exon 20 T790M and C797S mutations using ICE COLD-PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1390.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma

BackgroundUntil now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through...

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

Application of CO-amplification at Lower Denaturation temperature (COLD) PCR principle to DNA profiling by STR genotyping

Application of CO-amplification at Lower Denaturation temperature (COLD) PCR principle to DNA profiling by STR genotyping

COLD-PCR protocols to enrich KRAS codon 61 and 146 mutations in heterogeneous tumor samples.

e22125 Background: About 40% of colorectal cancer patients show mutations on codon 12-13 of KRAS but other, less frequent and mutually exclusive mutations, were described. In Italy, treatments with Cetuximab or Panitumumab for metastatic colorectal cancer are granted only for patients with no mutations on RAS genes and analysis of codon 12,13,59,61,117,146 of KRAS and NRAS are requested before the treatment scheduling. Principal limitation of conventional PCR-based methods for mutation analysis is the inability to selectively amplify low percentages of somatic variants in an elevated background of wild-type sequences. CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) is a simple approach of PCR that selectively enriches minority alleles in a mixture of wild-type and mutant sequences. This method requires the definition of a Critical Temperature (Tc) for each Dna sequence, lower than the melting temperature, at which mismatched Dna heteroduplex (WT-mutated alleles) are preferentially denatured, promoting selective amplification of mutant sequences. Minor allele enrichment by COLD-PCR can be performed in association to different methods of variants detection: Sanger Sequencing, HRM analysis, Pyrosequencing Methods: Comparison of theoretical sensitivity achievable after PCR or COLD-PCR amplification was carried out analyzing serial dilution of mutated cell lines DNA into WT DNA (from 50% to 0.25%). To optimize the enrichment of minority alleles we developed a Full COLD-PCR protocol to analyze the Tm-increasing mutation (183 A>C) on KRAS codon 61 and a Fast COLD-PCR protocol to analyze the Tm-reducing mutation (436 G>A) on KRAS codon 146. Critical temperature was defined experimentally. Results: In KRAS codon 61 and 146 analysis, minimum detectable percentage of mutated allele in WT background was about 10% using conventional PCR followed by both Sanger sequencing and HRM. Conversely limit of sensitivity reached 0.5% using both detection methods after COLD-PCR Conclusions: Our experiments proved as COLD-PCR could be a rapid and economic approach, adaptable to investigate other somatic variants, to enrich very low percentages of mutant alleles in very heterogeneous tumor samples.

A noninvasive assay for monitoring renal allograft status

Transplant rejection is a serious complication, sometimes threatening life of the patient. Although recent development of the new generation of immunosuppressive drugs reduced the incidence of acute rejection in kidney transplantation, the absence of noninvasive biomarkers of the rejection does not allow often the optimization of a prompt antirejection therapy. Serum creatinine is the most widely used marker for allograft function, however, it is not sensitive and specific enough to detect acute rejection. Other biomarkers are even less valuable for this purpose. Histological examination of renal allograft biopsy still remains the golden standard for diagnosing acute renal allograft rejection. Therefore, there is a high demand for reliable biomarkers for noninvasive monitoring of renal allograft status. Examination of urine in renal transplant recipients provides a logical and readily accessible approach for this monitoring. The high potency biomarkers for kidney allograft monitoring are fragments of DNA in recipient urine that originated from renal allograft cells. Because of the difference in the genetic origin these DNA can be distinguished from recipient DNA. Quantitative analysis of donor’s DNA, derived from cells of renal allograft, in recipient’s urine might be a reliable predictive tool for the kidney transplant rejection. We developed an assay to quantitate donor DNA content in recipient urine. Application of the technique—coamplification at lower denaturation temperature-PCR (COLD-PCR) increased the abundance of donor DNA that usually presents in recipient urine in quantities that are out of the detection range. This assay has a potential for routine application in clinical practice after statistical validation and additional modifications.

The “COLD-PCR Approach” for Early and Cost-Effective Detection of Tyrosine Kinase Inhibitor Resistance Mutations in EGFR-Positive Non–Small Cell Lung Cancer

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report

Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

Reply to the article entitled “Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report” by Galbiati et al., Clin Chem Lab Med 2014;52(4):505–9

Article Reply to the article entitled “Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report” by Galbiati et al., Clin Chem Lab Med 2014;52(4):505–9 was published on July 1, 2014 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 52, issue 7).

Pattern of clinically relevant mutations in consecutive series of Russian colorectal cancer patients

One hundred and ninety-five consecutive surgically treated Russian colorectal cancer (CRC) patients were retrospectively analyzed for the presence of mutations in KRAS, NRAS, BRAF and PIK3CA genes as well as for the microsatellite instability status. Comparison between high-resolution melting analysis, co-amplification at lower denaturation temperature PCR, DNA sequencing and allele-specific PCR for the detection of KRAS codon 12/13 mutations revealed that none of these methods alone provided satisfactory results in 100 % of the analyzed cases; this experience supports the use of more than one mutation-detecting technique at least in some circumstances. KRAS codon 12/13 substitutions were detected in 70 (35.9 %) CRC cases. Other mutations in the RAS/RAF genes occurred in 22 (11.3 %) cases and included rare KRAS (n = 6), NRAS (n = 8) and BRAF (n = 8) alterations. 5 BRAF mutations affected codon 600, while the remaining 3 potentially functional substitutions were located in the position 594. Twenty-four (12.3 %) CRC cases carried mutations in the PIK3CA, and 18 of these tumors also contained activating alteration in the RAS/RAF genes (p = 0.007). Only 3 (1.5 %) CRC cases showed high-level microsatellite instability (MSI-H) as determined by a panel of mononucleotide markers. Overall, the distribution of potentially predictive mutations in Russian CRC cases is similar to the one observed in other patient series of European descent. Noticeable occurrence of D594G mutation in BRAF oncogene and low frequency of MSI-H may deserve specific attention.

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

PCR-based techniques for the detection of minority mutations

With the development of molecular biology and molecular genetics,more attention has been taken to the advanced techniques which can be used to detect minority mutations.In recent years,many techniques such as amplification refractory mutation system,PCR clamping,pyrophosphorolysis-activated polymerization,co-amplification at lower denaturation temperature-PCR and digital PCR have been developed,which will make the hereditary disease diagnosis,cancer detection,noninvasive antenatal diagnosis step into a new stage in the near future.This review presents an overview of the principles,applications,advantages and disadvantages of these techniques.(Chin J Lab Med,2013,36:40-45) Key words: DNA Mutation alanalysis; Polymerase chain reaction; Transition temperature

COLD-PCR and Innovative Microarray Substrates for Detecting and Genotyping MPL Exon 10 W515 Substitutions

Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.