Drop-In, Drop-Out Allele-Specific PCR: A Highly Sensitive, Single-Tube Method

2004 ◽  
Vol 28 (3) ◽  
pp. 171-174 ◽  
Author(s):  
Alice Alexander ◽  
Nivedita Subramanian ◽  
Joel N. Buxbaum ◽  
Daniel R. Jacobson
Keyword(s):  
Drop Out ◽  
Single Tube ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Allele Specific Pcr

Individual single tube genotyping and DNA pooling by allele-specific PCR to uncover associations of polymorphisms with complex diseases

2007 ◽  
Vol 376 (1-2) ◽  
pp. 155-162 ◽  
Author(s):  
Antonio Casado-Díaz ◽  
Rafael Cuenca-Acevedo ◽  
José Manuel Quesada ◽  
Gabriel Dorado
Keyword(s):  
Complex Diseases ◽  
Dna Pooling ◽  
Single Tube ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Single tube allele specific PCR: a low cost technique for molecular screening of sickle cell anaemia in Nigeria

2018 ◽  
Vol 18 (4) ◽  
pp. 995
Author(s):  
Emuejevoke T Toye ◽  
Guido Van Marle ◽  
Wendy Hutchins ◽  
Olayinka Abgabiaje ◽  
Joy Okpuzor Okpuzor
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anaemia ◽  
Low Cost ◽  
Molecular Screening ◽  
Single Tube ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Plasma Detection of JAK2 Mutation Is Not Suitable as a Clinical Test

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5231-5231
Author(s):  
Mohamed E Salama ◽  
Sabina Swierczek ◽  
Roberto H Nussenzveig ◽  
Kimberly Hickman ◽  
Andrew Wilson ◽  
...  
Keyword(s):  
Quantitative Assessment ◽  
Jak2 V617f ◽  
Progressive Increase ◽  
Clinical Test ◽  
Jak2 Mutation ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Over Time

Abstract The 1849G>T (V617F) JAK2 gene activating point mutation is identified in a substantial number of chronic myeloproliferative neoplasms (MPN) and the mutant allele burden correlates with some of the complications of MPN, justifying the introduction of quantitative JAK2 assays in clinical practice. Recent reports suggested a higher detection rate of the JAK2 mutation using plasma rather than by cell analysis and even suggested its use to detect zygosity. In this study we questioned the biologic basis of this suggestion and tested its validity by comparing JAK2 mutation allele burden in paired cell and plasma samples over time using a highly sensitive quantitative assay. Peripheral blood samples were collected from known patients with a positive JAK2 mutation as detected in their purified granulocytes. Separation of plasma and granulocytes (GNC) were carried on day 0,3,5,7,9,10 after sample collection. Quantitative assessment of JAK2 was performed on a total of 63 samples from 7 patients using allele-specific PCR amplification of JAK2-exon14 in genomic DNA isolated from the paired purified peripheral blood granulocytes and plasma samples. The detection of wild and mutant JAK2 alleles was achieved by highly sensitive and discriminatory allele-specific PCR utilizing allele specific primers that incorporate a mismatched nucleotide and synthetic ”locked nucleic acid” (Nussenzveig Expl Hemat.35: 32–38, 2007). Quantitative assessment of the sample is achieved by real-time monitoring amplification from each allele (in separate reactions). The values for each reaction are then used to determine the frequency of the T-allele relative to the G-allele in the sample and statistical calculations were performed using SAS software, Version 9.1 of the SAS System Copyright © 2002–2003 SAS Institute Inc, Cary, NC, USA. Quantitative measurements of JAK2 V617F mRNA in these samples were also analyzed. The JAK2 V617F range was (0.2–90%) in GNC and (0–88%) in plasma with no concordance of corresponding values in any patient at any given time point. We identified a progressive increase in plasma JAK2 V617F DNA values accompanied by a progressive reciprocal decrease of JAK2 V617F in paired GNC samples in all cases over time, suggesting preferential lysis of the GNC bearing the JAK2 V617F mutation. Similar data were obtained using in vitro expansion of JAK2 V617F polycythemia vera erythroid progenitors. A repeated-measures ANOVA was calculated to determine whether there was interaction between GNC vs. plasma and time. The rate of change in the value of JAK2 did indicate an interaction and was significantly different (p=.003) between GNC and plasma. In one patient, the JAK2 V617F was detected in GNC (0.7%) but not in plasma sample on day 0. We conclude that detection of JAK2 V617F in plasma is due to decreased viability of the JAK2 bearing cells, which leads to progressive increase in plasma detection. There is no correlation between JAK2 V617F allelic burden values in GNC and plasma at any given point. JAK2 quantitation in plasma is not suitable as a clinical test.

scholarly journals Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2V617F mutant allele detection

BMC Cancer ◽  
2013 ◽  
Vol 13 (1) ◽  
Author(s):  
Giada V Zapparoli ◽  
Robert N Jorissen ◽  
Chelsee A Hewitt ◽  
Michelle McBean ◽  
David A Westerman ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Allele Detection ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes

2000 ◽  
Vol 46 (10) ◽  
pp. 1540-1547 ◽  
Author(s):  
Gerard G Donohoe ◽  
Maija Laaksonen ◽  
Kari Pulkki ◽  
Tapani Rönnemaa ◽  
Veli Kairisto
Keyword(s):  
Accurate Determination ◽  
Hereditary Hemochromatosis ◽  
Pcr Amplification ◽  
Single Tube ◽  
Melting Temperatures ◽  
Specific Pcr ◽  
Pcr Products ◽  
Allele Specific ◽  
Dissociation Curves ◽  
Allele Specific Pcr

Abstract Background: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y. Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches. Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished. Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.

scholarly journals Highly-Sensitive Allele-Specific PCR Testing Identifies a Greater Prevalence of Transmitted HIV Drug Resistance in Japan

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e83150 ◽  
Author(s):  
Masako Nishizawa ◽  
Junko Hattori ◽  
Teiichiro Shiino ◽  
Tetsuro Matano ◽  
Walid Heneine ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Hiv Drug Resistance ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Sensitive Allele ◽  
Allele Specific Pcr

Interleukin-6 and interleukin-6 promoter polymorphism (−174) G>C in patients with spontaneous venous thromboembolism

2006 ◽  
Vol 95 (05) ◽  
pp. 802-806 ◽  
Author(s):  
Rainer Vormittag ◽  
Kety Hsieh ◽  
Alexandra Kaider ◽  
Erich Minar ◽  
Christine Bialonczyk ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Interleukin 6 ◽  
Gel Electrophoresis ◽  
Promoter Polymorphism ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
History Of ◽  
Allele Specific ◽  
Highly Correlated ◽  
Allele Specific Pcr

SummaryIncreased levels of interleukin-6 (IL-6) have been reported in patients with a history of venous thromboembolism (VTE); however, prospective studies did not confirm an association between inflammatory markers that are highly correlated with IL-6 and the risk of VTE. It was the aim of our study to investigate the association of IL-6 and its promoter polymorphism (−174) G>C with the risk of spontaneous VTE. IL-6 was measured in 128 patients with deep venous thrombosis (DVT, 70 w / 58 m),105 with pulmonary embolism (PE, 58w/ 47 m) and 122 healthy controls (60 w / 62 m) with a highly sensitive ELISA (Quantikine− HS Human IL-6 Immunoassay, RnDSystems®). The promoter polymorphism was determined by genotyping, allele specific PCR was followed by high resolution gel-electrophoresis. Median concentrations [interquartile ranges] were 2.37 [1.51–3.89] (pg/ ml) in patients with DVT, 2.83 [1.83–4.87] in those with PE and 2.51 [1.71–4.78] in controls (p = 0.6, p = 0.4). Hetero- or homozygous carriers of the C allele (71% in DVT, 67% in PE and 59% among controls) did not have higher IL-6 levels than homozygous carriers of the G allele (median 2.60 vs. 2.59 pg/ml, p = 0.7). In conclusion, we found no association of IL-6 and its promoter polymorphism (−174) G>C with the risk of spontaneous VTE.

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