Lower Denaturation Temperature PCR Research Articles

Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment

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. 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. 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. 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.

COLD-PCR Enrichment of Rare Cancer Mutations prior to Targeted Amplicon Resequencing

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.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis.

Use of BLOCker sequencing (blocking oligonucleotide cycle sequencing) after ice COLD-PCR for detection of K-RAS mutations.

e13547 Background: The epidermal growth factor receptor (EGFR) antagonists, cetuximab and panitumumab, can be effective in colorectal cancer treatment. However, activating K-RAS mutations in codons 12 and 13 are associated with a poor EGFR antagonist response. As these mutations are seen in ~40% of CRC, high sensitivity assays for detection of these mutations will aid in patient treatment and disease monitoring. Methods: Ice COLD-PCR (Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) uses a reference sequence oligonucleotide (RS-oligo) to enhance detection of all low level mutations during PCR. The RS-oligo is complementary to one wild-type strand, so only one wild-type strand but both mutant DNA strands are amplified. This results in linear WT sequence amplification but exponential mutant sequence amplification. Ice COLD-PCR methods used in these studies can detect 0.05 – 0.01% K-RAS mutant DNA. For low level mutation confirmation we added BLOCker-oligos to sequencing reactions to block wild-type and allow mutant DNA sequencing (BLOCker-Sequencing). For blocking to occur, additional hybridization and denaturing steps are added to the cycle sequencing program. This insures that the BLOCker-oligo:WT DNA remains intact but the BLOCker-oligo:Mutant DNA is denatured. A sequencing primer overlapping the BLOCker-oligo 5’ end anneals to the mutant strand that is subsequently extended. Results: BLOCker sequencing was used to confirm presence or absence of low level K-RAS mutations after Ice COLD-PCR. When a K-RAS mutation is present at 0.05%-0.01%, the mutant peak may appear as baseline noise. When BLOCker Sequencing of these samples is performed, the presence of a mutation at this low level is enhanced in the BLOCker Sequencing electropherogram. There is no enhancement if the sample does not contain a mutation. Conclusions: Following Ice COLD-PCR amplification, BLOCker sequencing was able to confirm the presence or absence of very low levels of K-RAS mutations in the starting samples. BLOCker Sequencing is a novel method to confirm low level mutation presence when there is a uncertain DNA sequencing electropherogram result following Sanger sequencing.

High sensitivity detection and robust enrichment for PIK3CA somatic mutations using ice COLD-PCR technology.

e13546 Background: High sensitivity detection of cancer biomarkers facilitates early diagnosis and provides insight for intervention including stratification for drug treatment and monitoring changes in tumor mutation profiles by analyzing DNA isolated from serum/plasma or circulating tumor cells. PIK3CA, encoding the PI3K p110α catalytic subunit, is mutated frequently in common cancers, including breast and colon carcinomas. 80% of these mutations are represented by three amino acid substitutions in the enzyme’s helical (E542K and E545K) or kinase (H1047R) domains. Methods: In this study, we have applied improved and complete enrichment CO-amplification at lower denaturation temperature PCR (Ice COLD-PCR) technology for high sensitivity detection and robust enrichment of any mutations present in a DNA sample. Ice-COLD-PCR preferentially amplifies low levels of any mutant DNA in a sample containing a vast excess of wild-type DNA. The use of a reference sequence oligonucleotide complimentary to one of the wild-type DNA strands results in linear amplification of one of the wild type strands while both mutant strands amplify exponentially during ice COLD- PCR. After Ice COLD-PCR, there is sufficient mutant allele enrichment to allow Sanger sequencing confirmation of the mutation. Results: The use of Ice COLD-PCR allows for the enrichment of mutations any of the key mutations in the PIK3CA gene that cannot be detected by standard PCR followed by Sanger sequencing methods. Initial limit of detection experiments indicate that the detection and sequence confirmation of mutations in PIK3CA more sensitive than standard PCR amplification or allele specific probe methodologies. Conclusions: After Ice COLD-PCR, low level PIK3CA mutant alleles are enriched over the wild-type allele such that simple sequence confirmation on multiple downstream systems such as Sanger Sequencing or allele-specific probe platforms is possible. Ice COLD-PCR amplification of PIK3CA and other genes requires only a standard thermocycler coupled to any downstream sequence analysis platform and therefore can be developed as a powerful, highly sensitive diagnostic assay.

COLD-PCR: improving the sensitivity of molecular diagnostics assays

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.

BRAFV600E detection in melanoma is highly improved by COLD-PCR

BackgroundThe BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. MethodsCOLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations. ResultsWe used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. ConclusionsCOLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.

Full COLD-PCR Protocol for Noninvasive Prenatal Diagnosis of Genetic Diseases

To the Editor: After the discovery of fetal DNA in maternal plasma, investigators reported different strategies for the noninvasive prenatal diagnosis of genetic diseases (1). Despite the advances in improving the analytical sensitivity of methods, distinguishing between fetal and maternal sequences remains very challenging, and the field of noninvasive prenatal diagnosis of genetic diseases has yet to attain a routine application in clinical diagnostics. An innovative strategy that has been reported is based on COLD-PCR (coamplification at a lower denaturation temperature PCR),1 which exploits melting temperature ( T m) differences between variant or mismatched sequences and wild-type sequences. This approach uses a critical denaturation temperature ( T c) lower than the T m to selectively amplify minority mutated alleles (2). We have developed assays for the identification of fetal paternally inherited mutations in maternal plasma. These assays use full COLD-PCR for the detection of IVSI.110 (G>A) and Cd39 (C>T) HBB (globin, beta) gene mutations that cause β-thalassemia. Full COLD-PCR is based on the generation of heteroduplexes between mutant and wild-type sequences. These heteroduplexes melt at lower temperatures than the wild-type homoduplexes. They are selectively denatured at the T c and then subsequently amplified. Given that both the fetal and maternal DNA content in maternal plasma has been demonstrated to vary from pregnancy to …

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAF(V600E) in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAF(V600E) in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAF (V600E)), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAF (V600E). Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures.

Abstract 2119: Very high sensitivity detection of K-RAS exon 2 mutations using fast COLD-PCR

Abstract Therapeutic agents such as cetuximab and panitumumab are epidermal growth factor receptor (EGFR) antagonists that can be effective in the treatment of colorectal cancer. However some 40% of these tumors have activating K-RAS exon 2 codon 12 and 13 mutations that have a strong association with poor response to EGFR antagonists. Attempts have been made to detect the presence of K-RAS exon 2 mutations in free circulating DNA from plasma/serum from colorectal cancer patients with K-RAS exon 2 mutations in their tumors. The small amount of circulating DNA in serum and the low sensitivity of the detection techniques used have resulted in sporadic success in establishing this correlation. Co-amplification at Lower Denaturation Temperature-PCR (COLD-PCR) is a novel form of PCR that preferentially amplifies mutant alleles in a mutant/wild-type mixture with wild-type alleles in vast excess. Fast COLD-PCR, one of two forms of the method, is applied to point mutations or insertion/deletions that lower the melting temperature (Tm) of the mutant allele. In Fast COLD-PCR, preferential amplification of a mutant allele is achieved by setting the PCR denaturation temperature during each cycle at the critical temperature (Tc) at which mutant DNA duplexes but not wild-type duplexes are denatured. COLD-PCR is normally carried out in a thermocycler with independent temperature control of each well, ensuring that Tc is precisely achieved. Well-to-well temperature variation of standard, bench thermocyclers is often not within the narrow range required to achieve efficient COLD-PCR mutation enrichment. We have developed a variation of the Fast COLD-PCR method that reduces the need for precise well-to-well temperature control. In this study we have taken advantage of the Tm lowering of most of the K-RAS exon 2 mutations in codons 12 and 13 (G>A or G>T), and have successfully applied our variation of Fast COLD-PCR to their enrichment. A plasmid-based model system was established to measure the Limit of Detection (LOD) of the K-RAS exon 2 codon 12 mutation G12S (GGT>GAT). Mixtures of two plasmids, one bearing a wild-type copy of exon 2 and the other the G12S mutation were prepared at ratios of mutant/wild-type ranging from 1:10 to 1:1,000 (0.1%). A total of 10,000 plasmid copies alone or combined with purified human serum DNA were enriched. Two rounds of PCR were performed: standard PCR producing a 218-bp amplicon followed by enrichment using nested Fast COLD-PCR at a Tc of 80.9 °C producing a 161-bp amplicon. SURVEYOR® Nuclease/WAVE® HPLC and Sanger DNA sequencing were used to identify the G>A mutation in the enriched PCR product. The LOD for the G12S mutation before enrichment was 1% by SURVEYOR/WAVE and 5% by sequencing. After Fast COLD-PCR enrichment, the LOD was <0.1% in the absence and <0.4% in the presence of serum with both detection methods. This represents detection of less than 1 mutant in 1000 wild-type K-RAS copies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2119.

Abstract 2115: Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products

Abstract Much effort has been put into the development of sophisticated technologies enabling the detection of clinically significant low-level tumor specific KRAS mutations. The constitutively active KRAS oncogene harbors a mutation hotspot in codon 12 and 13 of exon 2. These mutations are predictors of response to treatment with the monoclonal antibodies panitumumab and cetuximab targeting the epidermal growth factor receptor (EGFR). Therefore, it is crucial to develop robust and sensitive assays targeting these mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation containing amplicons based on the lower melting temperature of heteroduplexes versus homoduplexes. We have developed an assay based on real-time COLD-PCR and high-resolution melting (HRM) targeting codon 12 and 13 for simultaneous detection of the various mutations found in this mutation hotspot. By using mutation containing cell-lines serially diluted into wild-type DNA we have compared the sensitivity of HRM after standard PCR with the sensitivity of HRM after COLD-PCR. We have also analyzed 60 colorectal cancer samples for which KRAS mutation status previously has been evaluated by the DxS kit which is approved by the U.S. Food and Drug Administration. Replacing standard PCR by COLD-PCR increased the sensitivity of the assay by up to 50 fold depending on the specific mutation. Furthermore, we have obtained very similar results when using our COLD-PCR assay as we obtained when using the DxS kit and confirmed the HRM results with Sanger sequencing. In conclusion, applying COLD-PCR to HRM analysis is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2115.

COLD-PCR–Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations

Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature-PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples. Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6-8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced. Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons. COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.

Two-round coamplification at lower denaturation temperature-PCR (COLD-PCR)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma

Reliable identification of cancer-related mutations in TP53 is often problematic, as these mutations can be randomly distributed throughout numerous codons and their relative abundance in clinical samples can fall below the sensitivity limits of conventional sequencing. To ensure the highest sensitivity in mutation detection, we adapted the recently described coamplification at lower denaturation temperature-PCR (COLD-PCR) method to employ two consecutive rounds of COLD-PCR followed by Sanger sequencing. Using this highly sensitive approach we screened 48 nonmicrodissected lung adenocarcinoma samples for TP53 mutations. Twenty-four missense/frameshift TP53 mutations throughout exons 5 to 8 were identified in 23 out of 48 (48%) lung adenocarcinoma samples examined, including eight low-level mutations at an abundance of approximately 1 to 17%, most of which would have been missed using conventional methodologies. The identified alterations include two rare lung adenocarcinoma mutations, one of which is a "disruptive" mutation currently undocumented in the lung cancer mutation databases. A sample harboring a low-level mutation ( approximately 2% abundance) concurrently with a clonal mutation (80% abundance) revealed intratumoral TP53 mutation heterogeneity. The ability to identify and sequence low-level mutations in the absence of elaborate microdissection, via COLD-PCR-based Sanger sequencing, provides a platform for accurate mutation profiling in clinical specimens and the use of TP53 as a prognostic/predictive biomarker, evaluation of cancer risk, recurrence, and further understanding of cancer biology.

Noninvasive prenatal diagnosis of congenital adrenal hyperplasia in cell-free fetal DNA with COLD-PCR

OBJECTIVE: Cell-free fetal DNA(cffDNA) present in the maternal plasma during pregnancy offers a potential material for noninvasive prenatal diagnosis. However, only about 3-6% occupation rate in the total circulating cell-free DNA limits its clinical application. To overcome this defect, various methods have been developed to optimize the extraction of cffDNA. Here, we first performed prenatal diagnosis to determine a paternally inherited congenital adrenal hyperplasia(CAH) mutation (Q318X of CYP21B gene) in cffDNA by co-amplification at lower denaturation temperature PCR(COLD-PCR), which is used to amplify minor alleles selectively from mixtures of wild-type and mutation-containing sequences.

GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabraud's syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579-584], a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing and prenatal identification of fetal alleles in maternal blood.

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

PCR for detecting low-prevalence mutations

Coamplification at lower denaturation temperature PCR (COLD-PCR) may offer improved sensitivity over traditional PCR for identifying low-prevalence mutations that occur in some cancers and infectious diseases, as well as identifying fetal alleles in maternal blood.