A simple method to overcome the inhibitory effect of heparin on DNA amplification
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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.
Conclusion
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.
Keywords
Single nucleotide polymorphism Polymerase chain reaction HeparinNotes
Compliance with ethical standards
Funding sources
PVS: Melville Trust for Care and Cure of Cancer 1 year research fellowship, Tenovus small research grant E13/01, One Year Research Fellowship supported by Harold Bridges Request, MRC Clinical Research Training Fellowship MR/M004007/1.
LYO: Cancer Research UK Research Training Fellowship (C10195/A12996).
NG: Medical Research Council Grant MR/J00913X/1.
SMF: Medical Research Council Grant MR/K018647/1.
MGD: Cancer Research Programme Grant C348/A12076.
Conflict of interest
No potential conflicts of interest were disclosed.
References
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