title: A simple method to overcome the inhibitory effect of heparin on DNA amplification.
abstract: BACKGROUND
  Genetic material from large patient cohorts is increasingly central to translational genetic research. However, patient blood samples are a finite resource and their supply and storage are often dictated by clinical and not research protocols. Our experience supports difficulty in amplifying DNA from blood stored in herparin; a scenario that other researchers may have or will encounter. This technical note describes a number of simple steps that enable successful PCR amplification.
  
  
  METHODS
  DNA was extracted using the Illustra Nucleon Genomic DNA Extraction Kit. PCR amplification was attempted using a number of commercially available PCR mastermixes.
  
  
  RESULTS
  PCR DNA amplification failed using ReddyMix™ PCR Master Mix, Thermo-Start® (Thermo Scientific Inc. US) and ZymoTaq™ (Zymo research, US) PCR mastermixes, as demonstrated absence of products on gel electrophoresis. However, using the Invitrogen™ (Thermo Scientific Inc., US) Platinum® Taq DNA Polymerase, PCR products were identified on a 1% agarose gel for all samples. PCR products were cleaned with ExoSAP-IT® (Affymetrix Inc., US) and a sequencing reaction undertaken using a standard Big Dye protocol. Subsequent genotyping was successful for all samples for alleles at the CDH1 locus.
  
  
  CONCLUSIONS
  From our experience a standard phenol/chloroform purification and using the Invitrogen™ Platinum® Taq has enabled the amplification of whole blood samples taken into lithium heparin and stored frozen for up to a month. This simple method may enable investigators to utilise blood taken in lithium heparin for DNA extraction and amplification.
authors.affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Western General Hospital Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK. pvaughan-shaw@nhs.net.
authors.forename: P G
authors.initials: PG
authors.lastname: Vaughan-Shaw
authors.affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Western General Hospital Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: M
authors.initials: M
authors.lastname: Walker
authors.affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Western General Hospital Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: L
authors.initials: L
authors.lastname: Ooi
authors.affiliation: Chromatin Biology, MRC Human Genetics Unit, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: N
authors.initials: N
authors.lastname: Gilbert
authors.affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Western General Hospital Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: S M
authors.initials: SM
authors.lastname: Farrington
authors.affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Western General Hospital Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: M G
authors.initials: MG
authors.lastname: Dunlop
mesh_terms: D001769:Blood / Q000187:drug effects; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification*; D005784:Gene Amplification; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D006493:Heparin; D004247:DNA
keywords: Heparin; Polymerase chain reaction; Single nucleotide polymorphism