title: Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1 Department of Pharmacy, Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University , Changzhou, China .
authors.forename: Er-Ping
authors.initials: EP
authors.lastname: Liu
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: He
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Guan
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Zheng-Hong
authors.initials: ZH
authors.lastname: Qin
authors.affiliation: 2 Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science , Suzhou, China .
authors.forename: Wan-Ping
authors.initials: WP
authors.lastname: Sun
mesh_terms: D000483:Alleles; D001774:Blood Chemical Analysis / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9 / Q000235:genetics; D004247:DNA / Q000097:blood* / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D015894:Genome, Human; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D054735:Phosphorothioate Oligonucleotides / Q000097:blood; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D064417:Vitamin K Epoxide Reductases / Q000235:genetics
chemical_list: D017931:DNA Primers; D054735:Phosphorothioate Oligonucleotides; D004247:DNA; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase