scholarly journals Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Raffaela Barbano ◽  
Barbara Pasculli ◽  
Michelina Coco ◽  
Andrea Fontana ◽  
Massimiliano Copetti ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Fast Method ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Melanoma Patients ◽  
Braf V600 Mutation

scholarly journals Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Cristin Roma ◽  
Claudia Esposito ◽  
Anna Maria Rachiglio ◽  
Raffaella Pasquale ◽  
Alessia Iannaccone ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Response To Treatment ◽  
Egfr Mutations ◽  
Clinical Samples ◽  
Training Set ◽  
Similar Frequency ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Validation Set

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct=37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

scholarly journals Somatic mutation detection efficiency in EGFR: a comparison between High Resolution Melting Analysis and Sanger Sequencing

2020 ◽  
Author(s):  
Reenu Anne Joy ◽  
Sukrishna Kamalasanan Thelakkattusserry ◽  
Narendranath Vikkath ◽  
Renjitha Bhaskaran ◽  
Damodaran Vasudevan ◽  
...  
Keyword(s):  
High Resolution ◽  
Somatic Mutation ◽  
Sanger Sequencing ◽  
Mutation Detection ◽  
Detection Efficiency ◽  
Limit Of Detection ◽  
Tumor Tissues ◽  
Allele Specific ◽  
Somatic Mutation Detection ◽  
Allele Fraction

Abstract Background: High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer .Methods: The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing.Results: The limit of detection of minor allele fraction of exon 19 mutation was 1% with Sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100% and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. Conclusion: The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

scholarly journals Rapid BRAF mutation detection in melanoma patients by immunohistochemistry

2017 ◽  
Vol 4 (1) ◽  
pp. 1-5
Author(s):  
László Fülöp ◽  
Katalin Götzer ◽  
Erzsébet Csernák ◽  
Danyil Szergejevics Kuznyecov ◽  
Erika Tóth
Keyword(s):  
Braf Inhibitor ◽  
Pcr Amplification ◽  
Diagnostic Algorithm ◽  
Braf V600e ◽  
Wild Type ◽  
Braf Gene ◽  
Braf Mutations ◽  
Activating Mutation ◽  
Melanoma Patients ◽  
Braf V600 Mutation

The V600E mutation is the most common (~90%) activating mutation of the BRAF gene. BRAF mutations have been frequently investigated in melanoma, colorectal cancer and papillary thyroid carcinoma. The importance of the detection of BRAF mutations has been rising by the routine use of Braf inhibitor therapy. We evaluated the usefulness of the BRAF V600E mutation-specific monoclonal antibody (VE1) in metastatic melanoma patients. To confirm the results of immunohistochemistry (IHC), we used COBAS 4800 BRAF V600 mutation test and PCR amplification followed by Sanger sequencing.36 of 105 patients have wild-type BRAF gene, 64 have V600E mutation and 5 of 105 have V600K mutation. Predicting the mutation only by IHC using VE1 antibody, all 58 positively scored specimen were V600E mutant. The V600K, the wild-type patients and 7 patients from the V600E mutant group scored as negative. Thus the specificity is 100% and the positive predictive value is 1 of the IHC method. After processing our data we could establish a cheaper diagnostic algorithm for rapid detection ofBRAF mutation.

scholarly journals K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification

2000 ◽  
Vol 46 (12) ◽  
pp. 1929-1938 ◽  
Author(s):  
Simon J Clayton ◽  
Frank M Scott ◽  
Jill Walker ◽  
Kay Callaghan ◽  
Kemal Haque ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mutation Detection ◽  
Lung Tumor ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Real Time Quantitative Pcr ◽  
Allele Specific ◽  
Specific Amplification ◽  
Tumor Dna

Abstract Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMSTM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%. Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

scholarly journals Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 872
Author(s):  
Alexey S. Chubarov ◽  
Igor P. Oscorbin ◽  
Maxim L. Filipenko ◽  
Alexander A. Lomzov ◽  
Dmitrii V. Pyshnyi
Keyword(s):  
Kras Mutation ◽  
Mutation Detection ◽  
Synergetic Effect ◽  
Model Systems ◽  
Wild Type ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Mutational Status ◽  
Allele Specific ◽  
Pcr Method

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

scholarly journals Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Reenu Anne Joy ◽  
Sukrishna Kamalasanan Thelakkattusserry ◽  
Narendranath Vikkath ◽  
Renjitha Bhaskaran ◽  
Sajitha Krishnan ◽  
...  
Keyword(s):  
High Resolution ◽  
Somatic Mutation ◽  
Sanger Sequencing ◽  
Mutation Detection ◽  
Detection Efficiency ◽  
Limit Of Detection ◽  
Tumor Tissues ◽  
Allele Specific ◽  
Somatic Mutation Detection ◽  
Allele Fraction

Abstract Background High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer. Methods The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing. Results The limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. Conclusion The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

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