title: A novel method for whole blood PCR without pretreatment.
abstract: PCR is usually performed on purified DNA. However, the extraction of DNA from whole blood is time consuming and involves the risk of contamination at every step. Hence, it is desirable to amplify DNA directly from whole blood. Earlier, investigators tried to achieve this target by either pretreatment of whole blood samples with different agents or by altering the conventional thermal cyclic conditions. This would make the technique cumbersome and time consuming. Here, we describe a simple protocol to amplify DNA directly from whole blood without the need of pretreatment. PCR buffer system was optimized in the laboratory and Apolipoprotein B gene was used as a model for this experiment. 480 bp was the target site for amplification. Fresh whole blood samples were used both from healthy and diseased individuals (coronary artery disease patients). Successful amplification was achieved with 1 μl volume of whole blood and it was comparable to that of genomic DNA. No pretreatment of whole blood samples was required with the optimized buffer system. 3mM concentration of MgCl(2) was observed to be optimal and hence used in the reaction mixture. Amplification was relatively better with this buffer system as compared to that of commercially available PCR buffer. With the present technique, amplicon detection did not require the centrifugation/dilution of the PCR products which further saves time. Successful amplification was achieved in both the healthy and diseased blood samples, indicating the robustness of the technique as changed blood composition and presence of increased inhibitory molecules in the diseased state did not seem to affect the efficacy of the present technique. In conclusion, as compared to the existing protocols for whole blood PCR, the present technique is relatively novel, simple, requires minimal steps and eliminates the need for additional standardizations.
authors.affiliation: Department of Biochemistry, Government Medical College, Amritsar-143001, India. ritu_gmc@rediffmail.com
authors.forename: Ritu
authors.initials: R
authors.lastname: Sharma
authors.forename: Amardeep Singh
authors.initials: AS
authors.lastname: Virdi
authors.forename: Prabhjeet
authors.initials: P
authors.lastname: Singh
mesh_terms: D001055:Apolipoproteins B / Q000235:genetics; D001769:Blood; D016022:Case-Control Studies; D003324:Coronary Artery Disease / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001055:Apolipoproteins B; D004247:DNA