Merck
  • Home
  • Search Results
  • Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

Genetic testing and molecular biomarkers (2015-09-12)
Er-Ping Liu, Yan Wang, Xiao-Hui He, Jun-Jie Guan, Jin Wang, Zheng-Hong Qin, Wan-Ping Sun
ABSTRACT

Point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood. However, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency. Our purpose was to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers. At high Mg(2+) concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood. Among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate. Meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect. These findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(-1639) loci. With its high DNA amplification efficiency and tolerance of various blood conditions, Pfu DNA polymerase can be used in clinical laboratories to analyze SNPs in whole blood samples.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Uracil, Vetec, reagent grade, 98%
Sigma-Aldrich
Uracil, BioReagent, suitable for cell culture
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.9% trace metals basis
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Betaine, BioUltra, ≥99.0% (NT)
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride, suitable for insect cell culture, BioReagent, ≥97.0%
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Uracil, ≥99.0%
Sigma-Aldrich
Betaine, ≥98% (perchloric acid titration)
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~0.025 M in H2O
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
Betaine solution, 5 M, PCR Reagent
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Magnesium chloride solution, 0.1 M