FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

Amicarelli, Giulia; Shehi, Erlet; Makrigiorgos, G. Mike; Adlerstein, Daniel

doi:10.1093/nar/gkm809

Cited by 49 publications

(36 citation statements)

References 44 publications

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“…The novel method was introduced recently for genotyping of KRAS gene (23,24,25,26). The are several advantages of real-time PCR techniques: PCR is not infl uenced by non-specifi c amplifi cation, there is no post-PCR processing of products (high throughput, low contamination risk), amplifi cation can be monitored in real-time, ultra-rapid cycling (30 minutes to 2 hours), confi rmation of specifi c amplifi cation by melting point analysis.…”

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confidence: 99%

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Ugorcakova

Hlavatý²,

Novotna³

et al. 2012

BLL

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Abstract:Objectives: The determination of gene mutations is important for the diagnosis and prognosis of various gastrointestinal cancers. The aim of our study was to develop a new procedure for the analysis of KRAS gene mutation by application of the real-time PCR method. Background: The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. Methods:The real-time PCR based technique using hybridization probes for fi ve most frequently KRAS codon 12 mutations and WT specifi c peptide nucleic acid (PNA) was performed. Our multiplex detection system was tested in various DNA samples (tissue, bile, pancreatic juice) of patients with different diagnoses of gastrointestinal tract disease obtained by endoscopy and ERCP. Results: We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplifi cation of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confi rmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRAS G12D it was in 44.4 %, KRAS G12V in 22.2 %, KRAS G12S in 14.8 %, KRAS G12A in 14.8 % and KRAS G12C in 3.8 %. The sensitivity of the assays is 1x10 -5 . Conclusions: Advantages of this technique are rapidity, accuracy and it is generally easy to perform. This method can be adapted for synchronic detection of multiple mutations and after readjustment by other type mutation of KRAS gene may serve as useful clinical tool for analyzing point mutations in various clinical samples (Tab. 3, Fig. 3, Ref. 42). Full Text in PDF www.elis.sk.

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confidence: 99%

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Ugorcakova

Hlavatý²,

Novotna³

et al. 2012

BLL

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“…15 Most other detection methods reported in literature [16][17][18][19] also involve PCR. PCR amplification enables mutation detection from a very small amount of samples.…”

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confidence: 99%

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

et al. 2009

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KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 801C (vs 941C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by 44.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffinembedded tissue.

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“…Most of the growing evidence indicates that detecting KRAS mutations in the plasma-circulating DNA may provide useful information to the clinician in diagnosing and treating PA, NSCLC and colorectal carcinomas Shyamala et al, 2008;Dawood et al, 2010). Various methods have been described for the detection of KRAS gene mutations, such as Restriction Fragment Length Polymorphism polymerase chain reaction (PCR-RELF) (CHEN et al, 2004), mutagenic PCR analysis (Boldrini et al, 2004), pyrosequencing (Ogino et al, 2004), real-time PCR (Amicarelli et al, 2007) and Sanger sequencing. Currently, the gold standard for KRAS mutations detection is conventional PCR amplification followed by direct sequencing (Sanger sequencing) on the primer of downstream.…”

Section: Research Articlementioning

confidence: 99%

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

Zhou²,

Song³

et al. 2015

Asian Pacific Journal of Cancer Prevention

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Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

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FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

Cited by 49 publications

References 44 publications

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

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