scholarly journals High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC

2001 ◽  
Vol 47 (3) ◽  
pp. 548-555 ◽  
Author(s):  
Elke Schaeffeler ◽  
Thomas Lang ◽  
Ulrich M Zanger ◽  
Michel Eichelbaum ◽  
Matthias Schwab
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
False Negative ◽  
Rapid Screening ◽  
Tpmt Activity ◽  
Polymorphism Analysis ◽  
Specific Pcr ◽  
Restriction Fragment Polymorphism ◽  
Allele Specific ◽  
Tpmt Gene

Abstract Background: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC). Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR). Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals. Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.

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 Detection of „Hotspot Mutations in Catalytic Subunit of Phosphatidylinositol 3-Kinase (Pik3ca) by Allele-Specific Polymerase Chain Reaction

2014 ◽  
Vol 14 (2) ◽  
pp. 10-15 ◽  
Author(s):  
Mendelova A. ◽  
Holubekova V. ◽  
Zubor P. ◽  
Jezkova E. ◽  
Gemzova K. ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Direct Sequencing ◽  
Chain Reaction ◽  
Pik3ca Gene ◽  
Specific Polymerase Chain Reaction ◽  
Specific Pcr ◽  
Allele Specific ◽  
Hotspot Mutations ◽  
Polymerase Chain ◽  
Allele Specific Pcr

Abstract The phosphatidylinositol 3-kinases (PI3Ks) are a family of proteins involved in the regulation of cell survival, growth, metabolism, and glucose homeostasis. Increased PI3K activity is associated with many cancers. PIK3CA gene (encoding p110 , the catalytic subunit of PI3K) is commonly mutated in breast cancer. In our study we focused on the detection of “hotspot” mutations in exons 9 and 20 of the PIK3CA gene in paraffin-embedded tissue of patients with breast cancer. We optimized conditions of allele specific polymerase chain reaction (PCR) and we used direct sequencing to verify our results. Overall, three “hotspot” mutations in PIK3CA gene in paraffin-embadded tissue from breast cancer were detected by allele-specific PCR. All results were verified by direct sequencing of PCR products and we observed 100% agreement between those two methods. We confirmed that allele-specific PCR assay is low cost method usefull for accurate detection of PIK3CA mutations.

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 418-418
Author(s):  
Radhashree Maitra ◽  
Jay B. Nayak ◽  
Atrayee Basu-Mallick ◽  
Arjun Sood ◽  
Titto A Augustine ◽  
...  
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
Cost Effective ◽  
Rapid Screening ◽  
Accurate Identification ◽  
Multiple Snps ◽  
Fast Screening ◽  
Effective Manner ◽  
Multiplex Sequencing ◽  
Multiple Samples

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of > 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4261-4261
Author(s):  
Gonzalo Manrique ◽  
Roberta Bittencout ◽  
Verónica Pérez ◽  
Vanesa Sholl ◽  
Monica Cappetta ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Low Cost ◽  
Mutation Screening ◽  
Point Mutations ◽  
Kinase Domain ◽  
Rapid Screening ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Allele Specific

Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.

Development of a multiplex allele-specific PCR assay for rapid screening of Lactoferrin TATA box polymorphisms

2012 ◽  
Vol 22 (6) ◽  
pp. 1241-1244
Author(s):  
Reza Valadan ◽  
Hassan Sharifiyazdi ◽  
Abdolah Mirzaei ◽  
Keshvad Hedayatian
Keyword(s):  
Tata Box ◽  
Rapid Screening ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Allele Specific ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

scholarly journals Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Sollip Kim ◽  
Hye Won Lee ◽  
Woochang Lee ◽  
Sail Chun ◽  
Won-Ki Min
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Sequencing ◽  
Reference Method ◽  
Korean Population ◽  
Clinical Samples ◽  
Tpmt Activity ◽  
Bone Marrow Toxicity ◽  
Thiopurine Methyltransferase ◽  
Allele Specific

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered.TPMTgenotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population.Materials and Methods. Three majorTPMTsingle-nucleotide polymorphisms (TPMT*2, *3B, and *3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing.Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency ofTPMT*3Cwas 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. NoTPMT*2, *3A, or *3Balleles were observed in this Korean population.Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

scholarly journals Allele-specific PCR assay to genotype SNP rs7903146 in TCF7L2 gene for rapid screening of diabetes susceptibility

2008 ◽  
Vol 52 (8) ◽  
pp. 1362-1366 ◽  
Author(s):  
Ludmila Alves Sanches Dutra ◽  
Patrícia Godoy Garcia Costa ◽  
Lara Franciele Ribeiro Velasco ◽  
Angélica Amorim Amato ◽  
Gustavo Barcelos Barra
Keyword(s):  
Rapid Screening ◽  
Complete Agreement ◽  
Diabetes Susceptibility ◽  
Specific Pcr ◽  
Allele Specific ◽  
Reverse Primer ◽  
Specific Amplification ◽  
Common Reverse Primer ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

OBJECTIVE: The aim of the present study is to validate a rapid and simple allele-specific PCR that genotypes TCF7/L2 rs7903146 (C/T) polymorphism with standard PCR instruments. METHODS: Two forward primers with variations in their 3' nucleotides were designed in such a way that each was specific for one of the two variants. They were combined with a common reverse primer into two PCR reactions. Specific amplification indicates the presence of the allele. One hundred and four DNA samples were genotyped by this method. To evaluate the assay, the polymorphism spanning region of 63 DNA samples representing the three possible genotypes was sequenced. RESULTS: The rs7903146 allele assignments derived from the allele-specific PCR were in complete agreement with sequencing. CONCLUSIONS: The assay described here is a suitable strategy for the TCF7/L2 rs7903146 (C/T) genotyping also allowing rapid and reliable identification.

scholarly journals Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears

2003 ◽  
Vol 47 (7) ◽  
pp. 2231-2235 ◽  
Author(s):  
Igor Mokrousov ◽  
Tatiana Otten ◽  
Boris Vyshnevskiy ◽  
Olga Narvskaya
Keyword(s):  
Mycobacterium Tuberculosis ◽  
False Negative ◽  
Direct Analysis ◽  
Multidrug Resistant ◽  
Single Step ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Mdr Tb ◽  
Allele Specific ◽  
Allele Specific Pcr

ABSTRACT We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.

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