Apr 24, 2012

A novel method for whole blood PCR without pretreatment

Gene
Ritu SharmaPrabhjeet Singh

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 commercial...Continue Reading

  • References8
  • Citations6

Mentioned in this Paper

Pathologic Cytolysis
Dimethyl Sulfoxide
Whole Blood
Coronary Artery Disease
Apolipoproteins B
Formamide
Hyperkalemic Periodic Paralysis
Gene Amplification Technique
Centrifugation
Coronary Arteriosclerosis

Related Feeds

Bacterial Pneumonia

Bacterial pneumonia is a prevalent and costly infection that is a significant cause of morbidity and mortality in patients of all ages. Here is the latest research.

Bacterial Pneumonia (ASM)

Bacterial pneumonia is a prevalent and costly infection that is a significant cause of morbidity and mortality in patients of all ages. Here is the latest research.