scholarly journals Detection of rare point mutation via allele-specific amplification in emulsion PCR

BMB Reports ◽  
2013 ◽  
Vol 46 (5) ◽  
pp. 270-275 ◽  
Author(s):  
Changming Cheng ◽  
Yin Zhou ◽  
Chao Yang ◽  
Juan Chen ◽  
Jie Wang ◽  
...  
Keyword(s):  
Point Mutation ◽  
Emulsion Pcr ◽  
Allele Specific ◽  
Specific Amplification

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.

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.

scholarly journals Epidermal Growth Factor rs4444903 A>G Gene Polymorphism Association with Chronic Hepatitis B Liver Disease Progression among Egyptian Children: A Multicenter Study

2021 ◽  
Vol 11 (1) ◽  
pp. 63-68
Author(s):  
Amal A. Mohamed ◽  
Gehan L.A. Hakeem ◽  
Gihan M. Babrs ◽  
Laila E. Abolfotoh ◽  
Nageh M. Shehata ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
Genotypic Frequency ◽  
Liver Disease Progression ◽  
Egyptian Children ◽  
Chronic Hepatitis B Virus ◽  
Genes Encoding ◽  
Allele Specific ◽  
Specific Amplification

Background: Polymorphisms of genes encoding the pro-inflammatory and anti-inflammatory cytokines can affect the clinical presentation of the infection. We aimed to assess the role of EGF gene single-nucleotide polymorphism in the outcome of chronic hepatitis B virus (HBV) infection in children. Methods: One hundred HBV-infected children and 75 healthy matched controls were enrolled in this prospective study. Patients included 18 chronic inactive and 82 chronic active carriers. EGF rs4444903 A>G genotypes were determined using allele-specific amplification. Results: Significant differences regarding EGF genotypic frequency (p=0.001) in patients compared to controls (p=0.001). Eighteen percent were inactive, and 82% were active carriers. AA, AG and GG genotypic frequency were 66.7%, 33.3%, 0% and were 3.7%, 37.8% and 58.5% in the inactive and active carriers, respectively, with significant differences regarding AA, AG, GG genotypic frequency (p=0.001 for all). EGF AA, AG, GG genotypes frequency were 1.9%, 33.3%, and 64.8%, respectively, with significant differences between cirrhotic and non-cirrhotic patients regarding AA, AG, GG genotypic frequency (p=0.001 for all). Conclusion: Increased G allele frequency in EGF rs4444903 A > G polymorphism in HBV- Egyptian children is associated with worse outcomes.

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