A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer

2021 ◽  
Author(s):  
Fatemeh Sheikhsofla ◽  
Behzad Poopak ◽  
Sajjad Firuzyar ◽  
Fatemeh Roudbari ◽  
Mojtaba Ghadiany
Keyword(s):  
Colorectal Cancer ◽  
Gene Mutations ◽  
Dot Blot ◽  
Braf Mutations ◽  
Kras Gene ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Reverse Dot Blot

Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods. Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively. Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D  and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.  Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

Analysis of BRAF and KRAS mutations in colorectal cancer and rectal carcinogenesis via fluorescent allele-specific PCR.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 436-436
Author(s):  
D. G. Stover ◽  
A. M. Cushman-Vokoun ◽  
C. L. Vnencak-Jones ◽  
J. Berlin
Keyword(s):  
Colorectal Cancer ◽  
Mapk Signaling ◽  
Braf V600e Mutation ◽  
Response To Therapy ◽  
Braf V600e ◽  
Braf Mutations ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

436 Background: Molecular analysis has become increasingly relevant in the evaluation of colorectal carcinomas. Mutations in Ras-MAPK pathway proteins KRAS (present in 30-40% of colorectal cancer) and BRAF (10-14% of colorectal cancer) or mismatch repair (MMR) enzymes can impact response to therapy and/or can assist in defining hereditary predisposition. High frequency microsatellite instability (MSI) in colorectal cancer is associated with improved outcomes. Targeted therapies against BRAF and other components of the Ras-MAPK signaling pathway are becoming important aspects of treatment in colorectal and other cancers. Methods: A retrospective study was performed on 111 paraffin-embedded tumor specimens submitted between 1/07 and 3/09 for MSI testing based on family history and/or histologic features. DNA samples were screened for the BRAF V600E mutation and 7 KRAS mutations in codons 12 and 13 using fluorescent allele specific PCR with capillary electrophoresis. Clinical data was collected via chart review. Results: 58 males and 53 females were studied. The incidence of KRAS and BRAF mutations among the 111 samples was 49.5% and 6.3%, respectively. KRAS G12D and G12V were the most common mutations, representing 57% of total KRAS mutations. Dually positive KRAS and MSI tumors exclusively demonstrated G12D and G13D mutations. KRAS and BRAF mutations were mutually exclusive. Rectal cancers did not show evidence of BRAF V600E mutation (p=0.04). KRAS, BRAF, and MSI status did not correlate with survival, however pre-operative and post-operative carcinoembryonic antigen (CEA) levels were significantly associated with survival both in univariate and multivariate analyses. Conclusions: We demonstrate that BRAF V600E mutation is significantly associated with colon cancer and not rectal cancer, suggesting that BRAF mutations are not relevant in rectal carcinogenesis. Correlation between specific KRAS mutation and outcome may require larger populations. No significant financial relationships to disclose.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

CLINICAL CHARACTERISTICS OF COLORECTAL CANCER IN PATIENTS WITH EGFR-SIGNALING PATHWAY GENE MUTATIONS

2019 ◽  
pp. 60-67
Author(s):  
I.A. Bogomolova ◽  
I.I. Antoneeva ◽  
D.R. Dolgova
Keyword(s):  
Colorectal Cancer ◽  
Adjuvant Chemotherapy ◽  
Clinical Characteristics ◽  
Gene Mutations ◽  
Egfr Signaling ◽  
Braf Gene ◽  
Braf Mutations ◽  
Kras Gene ◽  
Egfr Signaling Pathway ◽  
Gene Exon

KRAS, NRAS, BRAF mutations are associated with an unfavorable prognosis for colorectal cancer (CRC). At the same time, there is no single point of view on disease development during adjuvant chemotherapy (ACT). Objective. The authors aimed at studying KRAS, NRAS and BRAF mutations in the tumor and their influence on the clinical characteristics of colorectal cancer development. Materials and Methods. Paraffin blocks of the primary CRC tumor (n=37) were used as the material for the study. Using genomic DNA, isolated from the primary tumor, real-time PCR was used to determine the most common mutations in CRC: KRAS gene (exon 2, codon region 12–13), NRAS gene (exon 3, codon region 61), B6F V600E gene. Results. The results of genotyping of DNA samples isolated from the primary CRC tumor paraffin blocks showed that BRAF gene mutations were detected in 8.2 % of cases, NRAS gene mutations were detected in 5.4 % of cases, and KRAS gene mutations were detected in 37.8 % of cases. The authors didn’t reveal any dependencies of the mutation distribution on patients’ gender and age. The examined mutations were more common in adenocarcinomas of high and moderate degrees of differentiation. The relapse-free period after ACT in patients with identified KRAS, NRAS, BRAF gene mutations is significantly less than in those without mutations. Conclusion. The findings suggest that EGFR signaling pathway mutations (KRAS, NRAS and BRAF) increase the risk of disease recurrence and are an unfavorable prognostic factor. Keywords: colorectal cancer, NRAS, KRAS, BRAF mutations, adjuvant chemotherapy.

Comparison of PCR-RFLP with Allele-Specific PCR in Genetic Testing for Spinal Muscular Atrophy

2003 ◽  
Vol 7 (4) ◽  
pp. 277-281 ◽  
Author(s):  
Ruliang Xu ◽  
Shuji Ogino ◽  
Va Lip ◽  
Hong Fang ◽  
Bai-Lin Wu
Keyword(s):  
Genetic Testing ◽  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Allele Specific Pcr

Analysis ofIGF2 gene imprinting in breast and colorectal cancer by allele specific-PCR

1999 ◽  
Vol 187 (5) ◽  
pp. 518-522 ◽  
Author(s):  
Kankatsu Yun ◽  
Hidenobu Soejima ◽  
Arend E. H. Merrie ◽  
John L. McCall ◽  
Anthony E. Reeve
Keyword(s):  
Colorectal Cancer ◽  
Gene Imprinting ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Rapid characterization of b-thalassemia mutations by reverse dot blot and allele-specific pcr analysis

2002 ◽  
Vol 21 (3) ◽  
pp. 283-286 ◽  
Author(s):  
Sonja Pavlovic ◽  
Jelena Urosevic ◽  
Tatjana Djureinovic ◽  
Dragana Janic ◽  
Lidija Krivokapic-Dokmanovic
Keyword(s):  
Pcr Analysis ◽  
Amplification Refractory Mutation System ◽  
Thalassaemia Major ◽  
Dot Blot ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
The Republic ◽  
Rapid Characterization ◽  
Reverse Dot Blot

This paper reports a case of b-thalassaemia major whose molecular diagnosis was achieved by using modern methods of molecular genetics. This example demonstrates the strategy we chose to detect b-thalassaemia mutations in the Republic of Serbia in order to complete molecular screening in our population and to make prenatal diagnosis in pregnancies at risk. The analysis of genomic DNA isolated from the blood of patient affected with thalassaemia major is carried out by the methods: RDB (reverse dot blot) and ARMS (amplification refractory mutation system). It is shown that the patient is a compound heterozygote for two b- -thalassaemic mutations: b+IVSI-110 and b+IVSI-6.

scholarly journals Limits of Dideoxysequencing in the Detection of Somatic Mutations in Gastrointestinal Stromal Tumors

2015 ◽  
Vol 15 (3) ◽  
pp. 13-20 ◽  
Author(s):  
K Jasek ◽  
V Buzalkova ◽  
P Szepe ◽  
L Plank ◽  
Z Lasabova
Keyword(s):  
Gastrointestinal Stromal Tumors ◽  
Somatic Mutations ◽  
Low Cost ◽  
Gene Mutations ◽  
Wild Type ◽  
Stromal Tumors ◽  
Specific Pcr ◽  
Allele Specific ◽  
Detection Of Mutations ◽  
Allele Specific Pcr

Abstract Detection of mutations in cancer is particularly important in terms of proper treatment and targeted therapy. The aim of this study was the comparison of two methods: allele-specific PCR (AS-PCR) and dideoxysequencing applied for the identification of BRAF gene mutations in wild-type gastrointestinal stromal tumors (WT GISTs). We have optimized the conditions for the detection V600E mutation representing the c.1799 T>A substitution by AS-PCR and have used dideoxysequencing to verify our results. In nine cases, we were able to detect the mutation by AS-PCR approach; however, the mutations have been confirmed by dideoxysequencing in four cases only. AS-PCR is fast and low cost method for the detection of V600E mutation which was validated as a sensitive assay for the identification of the most common BRAF mutation in DNA extracted from paraffin-embedded tissue of WT GISTs.

Sign in / Sign up

Export Citation Format

Share Document