Clinical Applications of Whole-Blood PCR with Real-Time Instrumentation

Abstract Background: As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR). Methods: We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prism™ 7700 sequence detection system with minor groove– binding nonfluorescent quencher probes. Results: We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method. Conclusion: This approach has applications for testing other medically relevant single-nucleotide polymorphisms.

Download Full-text

Single Nucleotide Polymorphisms in the Chicken Mx gene at Position 2032 by Real-time Allele-specific PCR Melting-curve Analysis

The Journal of Poultry Science ◽

10.2141/jpsa.009070 ◽

2010 ◽

Vol 47 (2) ◽

pp. 133-138 ◽

Cited By ~ 1

Author(s):

Xiangqun Ye ◽

Zongcheng Tan ◽

Yueling Zhang ◽

Kangsheng Li

Keyword(s):

Single Nucleotide Polymorphisms ◽

Real Time ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

Download Full-text

The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method

The Analyst ◽

10.1039/c8an00116b ◽

2018 ◽

Vol 143 (14) ◽

pp. 3292-3301 ◽

Cited By ~ 9

Author(s):

Huihui Mao ◽

Guanghua Luo ◽

Yuxia Zhan ◽

Jun Zhang ◽

Shuang Yao ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

Curve Analysis ◽

Probe Method ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide

The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve analysis, which might require only one pair of primers and one probe.

Download Full-text

A Simple Genetic Thrombosis Score Of Five Single Nucleotide Polymorphisms Is Associated With Risk Of First Venous Thrombosis In Pregnant Women

Blood ◽

10.1182/blood.v122.21.3617.3617 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3617-3617

Author(s):

Lance A. Bare ◽

Hugoline G. de Haan ◽

Andre R. Arellano ◽

Carmen H. Tong ◽

James J. Devlin ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Venous Thrombosis ◽

Pregnant Women ◽

Odds Ratio ◽

Factor V Leiden ◽

Postpartum Women ◽

Factor V ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Unit Increase

Abstract Background Venous thrombosis (VT), a major cause of maternal morbidity and mortality, is increased 4- to 5- fold during pregnancy. A thrombosis score comprising 5 single nucleotide polymorphisms (SNPs) [rs6025 (Factor V Leiden), rs1799963 (Prothrombin 20210 G>A), rs8176719 (ABO 261G>deletion), rs2066865 (FGG 10034 C>T) and rs2036914 (F11 10364T>C)] was previously shown to be associated with VT events in the general population. We asked whether this thrombosis score could predict VT in pregnant and postpartum women in a case-control study on the etiology of thrombosis. Methods We studied women during pregnancy and up to three months postpartum (55 controls, 144 cases) selected from over 5000 female cases and controls of the MEGA (the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis) study. The thrombosis score was calculated for each individual as a sum of risk alleles weighted by the log of the published odds ratio (de Haan et al, Blood 2012). The VT risk per unit of thrombosis score was calculated as a continuous variable in a logistic regression model that adjusted for age, smoking history and family history of VT. The thrombosis scores ranged from 0 to 1.8 in female participants in MEGA with genotypes for the 5 SNPs (n=3,464). Results In pregnant and postpartum women, the odds ratio per unit increase in thrombosis score was 10.7 (95%CI 3.2 to 36.2). When the thrombosis score was evaluated in a sub study of pregnant and postpartum women who do not carry factor V Leiden—an important contributant to the risk score— the remaining 4-SNP thrombosis score was also associated with VT: the odds ratio per unit increase in thrombosis score was 10.6 (95%CI 2.3-48.3).The American College of Obstetricians and Gynecologists recommends thromboprophylaxis during pregnancy and postpartum periods in women who are compound carriers of both factor V Leiden and prothrombin 20210 G>A; these compound carriers who do not carry other risk variants would have a thrombosis score of 1.02. Compared with women in the bottom quintile of thrombosis scores, women with thrombosis scores equal to or greater than 1.02 had an odds ratio for VT of 12.3 (95%CI 1.5 to 99.9). About 2% of female controls in MEGA had thrombosis scores equal to or greater than 1.02. Conclusions A 5-SNP thrombosis score that combines the VT risk of 5 genetic variants is associated with VT in pregnant women. Disclosures: Bare: Celera: Employment. Arellano:Celera: Employment. Tong:Celera: Employment. Devlin:Celera: Employment.

Download Full-text

Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System

Clinical Chemistry ◽

10.1373/49.10.1599 ◽

2003 ◽

Vol 49 (10) ◽

pp. 1599-1607 ◽

Cited By ~ 13

Author(s):

Sha-Sha Wang ◽

Keith Thornton ◽

Andrew M Kuhn ◽

James G Nadeau ◽

Tobin J Hellyer

Keyword(s):

Single Nucleotide Polymorphisms ◽

Dna Sequencing ◽

Real Time ◽

Whole Blood ◽

Nucleotide Polymorphisms ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Single Nucleotide ◽

Buccal Swabs ◽

Real Time Detection

Abstract Background: The BD ProbeTec™ ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human β2-adrenergic receptor (β2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 μL of sample per reaction. All six β2AR assays were able to accommodate ≥20 μL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six β2AR assays agreed with DNA sequencing results. Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Download Full-text