A novel method for whole blood PCR without pretreatment
Gene 501 (2012) 85–88
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A novel method for whole blood PCR without pretreatment
Ritu Sharma a,⁎, Amardeep Singh Virdi b, Prabhjeet Singh b a b
Department of Biochemistry, Government Medical College, Amritsar-143001, India
Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, India
a r t i c l e
i n f o
Article history:
Accepted 22 March 2012
Available online 11 April 2012
Keywords:
DNA
Whole blood
Amplification
PCR
Coronary artery disease
Apolipoprotein B gene
a b s t r a c t
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. 3 mM concentration of MgCl2 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
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A novel method for whole blood PCR without pretreatment
Ritu Sharma a,⁎, Amardeep Singh Virdi b, Prabhjeet Singh b a b
Department of Biochemistry, Government Medical College, Amritsar-143001, India
Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, India
a r t i c l e
i n f o
Article history:
Accepted 22 March 2012
Available online 11 April 2012
Keywords:
DNA
Whole blood
Amplification
PCR
Coronary artery disease
Apolipoprotein B gene
a b s t r a c t
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. 3 mM concentration of MgCl2 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
References: Alison, C., Melinda, H., John, I., Cyril, M., David, S., Frank, C., 2005. Clinical applications of whole-blood PCR with real-time instrumentation Burckhardt, J., 1994. Amplification of DNA from whole blood. PCR Methods Appl. 3, 239–243. de Vries, Johan E., Petal Wijme AHM, Karly H, van Dieijen-Visser, M.P., Otto B, 2001. Chem. 47, 1701–1702. McCuskar, J., Dawson, M.T., Noone, D., Gannon, F., Smith, T., 1992. Improved method for direct amplification from whole blood Jadaon, M.M., Dashti, A.A., Lewis, H.L., Habeeb, F.M., 2009. Whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method Mercier, B., Gaucher, C., Feugeas, O., Mazurier, C., 1990. Direct PCR from whole blood, without DNA extraction Misra, A., Nishanth, S., Pasha, S.T., Pandey, R.M., Sethi, P., Rawat, D.S., 2001. Relationship of Xba1 and EcoR1 polymorphisms of apolipoprotein-B gene to dyslipidemia and Narayan, S., 1992. Overview of principles and current uses of DNA probes in clinical and laboratory medicine Nordvag, B.Y., Husby, G., Raafat el- Gewely, M., 1992. Direct PCR of washed blood cells. Panacchio, M., Lew, A., 1991. PCR in the presence of 8% (v/v) blood. Nucleic Acids Res. 19, 1151. Rudbeck, L., Dissing, J., 1998. Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR Sharma, R., Mahajan, M., Singh, B., Singh, G., Singh, P., 2011. Role of the APOB gene polymorphism (c.12669G>A, p.Gln4154Lys) in coronary artery disease in the Indian Punjabi population Shoulders, C.C., et al., 1985. Molecular cloning of human LDL apolipoprotein B cDNA: evidence for more than one year per haploid genome Victor, S.T., et al., 2009. Isothermal single nucleotide polymorphism genotyping and direct PCR from whole blood using a novel whole-blood lysis buffer. Mol. Diagn. Zhang, Y., Isaacman, D.J., WadowskyRM, White J.R., Post, J.C., Ehrlich, G.D., 1995. Detection of streptococcus pneumonia in whole blood by PCR. J. Clin. Microbiol. 33, 596–601. Zhang, Z., Kermekchiev, M.B., Barnes, W.M., 2010. Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq