scholarly journals Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000

BioTechniques ◽  
2008 ◽  
Vol 44 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Jonci N. Wolff ◽  
Neil J. Gemmell
Keyword(s):  
Real Time ◽  
Fluorescent Probes ◽  
Real Time Pcr ◽  
Allele Specific

Allele specific fluorescent probes and real time PCR for HLA typing

2002 ◽  
Vol 63 (10) ◽  
pp. S6
Author(s):  
Alison Castley ◽  
S.L Castley ◽  
David C Sayer ◽  
Frank T Christiansen
Keyword(s):  
Real Time ◽  
Fluorescent Probes ◽  
Real Time Pcr ◽  
Hla Typing ◽  
Allele Specific

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

2011 ◽  
Vol 49 (9) ◽  
pp. 3168-3174 ◽  
Author(s):  
Andrew S. Bae ◽  
Karin S. Ku ◽  
Michael D. Miller ◽  
Hongmei Mo ◽  
Evguenia S. Svarovskaia
Keyword(s):  
Hepatitis C ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Genotype 1A ◽  
Allele Specific

scholarly journals A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Qian Wang ◽  
Dimitrios P. Kontoyiannis ◽  
Ruoyu Li ◽  
Wei Chen ◽  
Dingfang Bu ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Gene Mutations ◽  
Health Concern ◽  
Content Type ◽  
Clinical Strains ◽  
Allele Specific ◽  
Resistant Strains

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

scholarly journals Rapid and Reliable Method for Cytochrome P450 2D6 Genotyping

2002 ◽  
Vol 48 (9) ◽  
pp. 1412-1417 ◽  
Author(s):  
Ulrike M Stamer ◽  
Bettina Bayerer ◽  
Stephanie Wolf ◽  
Andreas Hoeft ◽  
Frank Stüber
Keyword(s):  
Melting Point ◽  
Real Time ◽  
Real Time Pcr ◽  
Reliable Method ◽  
Cytochrome P450 2D6 ◽  
Cyp2d6 Gene ◽  
Melting Point Analysis ◽  
Point Analysis ◽  
Allele Specific ◽  
P450 2D6

Abstract Background: Single-nucleotide polymorphisms and single-base deletions within the cytochrome P450 2D6 (CYP2D6) gene have been associated with a poor metabolizer (PM) phenotype and display a frequency of 7–10% in the Caucasian population. Methods: We developed a reliable and rapid procedure to identify five major PM-associated mutations (CYP2D6*4, *7, and *8) and deletions (CYP2D6*3 and *6) by real-time PCR with subsequent fluorometric melting point analysis of the PCR product. These polymorphisms within the CYP2D6 gene were detected by use of two primer pairs and five different pairs of hybridization probes. DNA extracted from whole blood of 323 individuals was analyzed, and results were compared with genotypes obtained by allele-specific multiplex PCR. In case of uncertain results, additional sequence analysis was performed. Results: Genotyping results by real-time PCR were 100% reliable, whereas conventional allele-specific multiplex PCR produced uncertain results for 12.1% of samples, as confirmed by sequence analysis. Costs for reagents and consumables were considerably higher for the real-time PCR technology, but labor time was reduced by 2 h compared with allele-specific PCR. The allele frequencies in the population investigated were 0.186 for allele *4, 0.026 for allele *5, 0.009 for allele *3, 0.031 for allele *6, and 0.002 for allele *8. The defective CYP2D6*7 allele was not found. In addition, three additional mutations were detected, one of them displaying a PM genotype. Conclusion: Genotyping of CYP2D6 by real-time PCR with fluorometric melting point analysis is a rapid and reliable method.

A fast and reliable method for detecting SNP rs67384697 (Hsa‐miR‐148a binding site) by a single run of allele‐specific real‐time PCR

HLA ◽  
2020 ◽  
Vol 96 (3) ◽  
pp. 312-322
Author(s):  
Mauro S. Malnati ◽  
Priscilla Biswas ◽  
Elisabetta Ugolotti ◽  
Eddi Di Marco ◽  
Francesca Sironi ◽  
...  
Keyword(s):  
Binding Site ◽  
Real Time ◽  
Real Time Pcr ◽  
Reliable Method ◽  
Allele Specific

BRCA1 gene mutations frequency estimation by allele-specific real-time PCR of pooled genomic DNA samples

The Breast ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 532-536 ◽  
Author(s):  
Maksim S. Anisimenko ◽  
Dmitriy V. Mitrofanov ◽  
Olga B. Chasovnikova ◽  
Mikhail I. Voevoda ◽  
Sergey P. Kovalenko
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Frequency Estimation ◽  
Gene Mutations ◽  
Brca1 Gene ◽  
Allele Specific

A multiplex allele-specific real-time PCR assay for screening of ESR1 mutations in metastatic breast cancer

2015 ◽  
Vol 98 (2) ◽  
pp. 152-157 ◽  
Author(s):  
Ting Wang ◽  
Jin-Hui Liu ◽  
Jie Zhang ◽  
Le Wang ◽  
Chao Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Real Time ◽  
Real Time Pcr ◽  
Metastatic Breast ◽  
Pcr Assay ◽  
Allele Specific ◽  
Esr1 Mutations
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