An Improved Protocol for High Quantity and Quality of Genomic DNA Isolation from Human Peripheral Blood

Main Article Content

Eric Tzyy Jiann Chong
Lucky Poh Wah Goh
Keh Kheng Png
Ping-Chin Lee*

Abstract

DNA isolation is the most essential step in molecular studies. The quantity and quality of the isolated DNA may subsequently influence the reliability and reproducibility of experimental data especially those involving downstream analysis such as polymerase chain reaction (PCR). In this study, we report an improved protocol for isolating high quantity and quality of genomic DNA from human peripheral blood that is as competitive to commercial kits. The concentration of the genomic DNA isolated using the improved protocol was >100 ng/µl and the A260/A280 absorbance ratio was ranged within 1.604-1.861. When the DNA integrity was measured using Fragment AnalyzerTM, the isolated genomic DNA was highly intact with a genomic quality number of ≥7.0. The isolated genomic DNA was adequate for further molecular analyses including standard PCR and real-time PCR. More importantly, the improved protocol is able to isolate the genomic DNA of Plasmodium parasites that infected human red blood cells, thus enabling them to be correctly identified up to the species level using multiplex PCR.


 


Keywords: DNA isolation; human blood; polymerase chain reaction; Plasmodium parasites


*Corresponding author: Tel.: (+6) 088320000 ext. 100101 Fax: (+6) 088435324


                                             E-mail: [email protected]

Downloads

Download data is not yet available.

Article Details

Section
Research Articles

References

[1] Lorenz, T.C., 2012. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments, 63, 3998, https://doi.org/10.3791/ 3998
[2] Holden, M.J., Blasic, J.R., Bussjaeger, L., Kao, C., Shokere, L.A., Kendall, D.C., Freese, L. and Jenkins, G.R., 2003. Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of tract amounts of biotechnology-derived DNA. Journal of Agriculture and Food Chemistry, 51, 2468-2474.
[3] Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonák, J., Lind, K., Sindelka, R., Sjöback, R., Sjögreen, B., Strömbom, L., Ståhlberg, A. and Zoric, N., 2006. The real-time polymerase chain reaction. Molecular Aspects of Medicine, 27, 95-125.
[4] Lee, J.H., Park, Y., Choi, J.R., Lee, E.K. and Kim, H.S., 2010 Comparisons of the three automated systems for genomic DNA extraction in a clinical diagnostic laboratory. Yonsei Medical Journal, 51, 104-110.
[5] Chong, E.T.J., Lee, C.C., Chua, K.H., Chuah, J.A. and Lee, P.-C., 2014. RsaI but not DraI polymorphism in CYP2E1 gene increases the risk of gastrointestinal cancer in Malaysians: a case-control study. BMJ Open, 4(1), https://doi.org/10.1136/bmjopen-2013-004109
[6] Chong, E.T.J., Abdul, A.N.F. and Lee, P.-C., 2018. Association of IRX3 rs3751723 polymorphism with the risk of overweight and obesity: case-control study and meta-analysis. Meta Gene, 16, 50-56.
[7] Desjardins, P. and Conklin, D., 2010. NanoDrop microvolume quantitation of nucleic acids. Journal of Visualized Experiments, 45, 2565, https://doi.org/10.3791/2565
[8] Fleige, S. and Pfaffl, M.W., 2006. RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine, 27, 126-139.
[9] Chew, C.H., Lim, Y.A.L., Lee, P.C., Mahmud, R. and Chua, K.H., 2012. Hexaplex PCR detection system for identification of five human Plasmodium species with an internal control. Journal of Clinical Microbiology, 50, 4012-4019.
[10] Goh, X.T., Lim, Y.A.L., Vythilingam, I., Chew, C.H., Lee, P.C., Ngui, R., Tan, T.C., Yap, N.J., Nissapatorn, V. and Chua, K.H., 2013. Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed by PlasmoNexTM. Malaria Journal, 12, 264, https://doi.org/10.1186/1475-2875-12-264
[11] William, T., Jelip, J., Menon, J., Anderios, F., Mohammad, R., Mohammad, T.A.A., Grigg, M.J., Yeo, T.W., Anstey, N.M. and Barber, B.E., 2014. Changing epidemiology of malaria in Sabah, Malaysia: increasing incidence of Plasmodium knowlesi. Malaria Journal, 13, 390, https://doi.org/10.1186/1475-2875-13-390
[12] Lee, P.C., Chong, E.T.J., Anderios, F., Lim, Y.A.L., Chew, C.H. and Chua, K.H., 2015. Molecular detection of human Plasmodium species in Sabah using PlasmoNexTM multiplex PCR and hydrolysis probes real-time PCR. Malaria Journal, 14, 28, https://doi.org/10.1186/s 12936-015-0542-5