Improvements of PCR Amplification of Guanine plus Cytosine-Rich Constructs of Mycobacterium tuberculosis Gene using DMSO
DOI:
https://doi.org/10.29303/jku.v1i1.45Abstract
Vaccine research entered a new era when several useful molecular research tools were established. Instead of attenuated virulent microorganisms or killed virulent microorganisms, effective subunit vaccines were developed using recombinant DNA technology. By using the technology, selected genes of the virulent microorganisms can be amplified, cloned, expressed, and evaluated as vaccine components in challenge studies. However, a major bottleneck with the amplification of functional genes from Mycobacterium tubeculosis containing guanine plus cytosin-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. To solve this problem in this research, the amplification reaction was modified by addition of dimethyl sulfoxide (DMSO) into amplification reaction mixtures. It was found that 10% (v/v) of DMSO in the reaction mixture improved the amplification of GC-rich template of M. tuberculosis gene. This result indicating that amplification of unbalanced content of G and C deoxyribonucleotides genome could be improved using low-cost organic molecule, DMSO. Therefore, the DMSO should be widely useful as an enhancer to improve the amplification of GC rich construct from other genome. Keywords: Mycobacterium tubeculosis,vaccine, dimethyl sulfoxide, Guanine-CytosineReferences
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Planning, Financing. 2007.
2. Moss,A.R., Alland,D., Telzak,E., Hewlett,D., Jr., Sharp,V.,Chiliade,P.,
LaBombardi,V., Kabus,D., Hanna,B.,Palumbo,L., Brudney,K., Weltman,A.,
Stoeckle,K., Chirgwin,K., Simberkoff,M.,32 Moghazeh,S., Eisner,W., Lutfey,M., and Kreiswirth,B. (1997) A city-wide outbreak of a multiple-drugresistant strain of M. tuberculosis in New York. Int.J.Tuberc.Lung Dis. 1, 115-121
3. Singh,J.A., Upshur,R., and Padayatchi,N. 2007. XDR-TB in South Africa: no time for denial or complacency. PLoS.Med. 4, e50
4. Colditz, GA, Brewer, TF., Nerkey CS., Wilson ME., Burdick E., Fineberg HV.,
and Mosteller F. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. JAMA 271; 689.
5. Ginsberg AM., 1998. The tuberculosis epidemic: scientific challenges and
oppurtunities. Public Health Rep. 113; 128.
6. Dale JW, and Patki. 1990. Mycobacterial gene expression and regulation. In: McFadden J (Ed) Molecular Biology of the Mycobacteria. Surrey University Press, London, United Kingdom.
7. Jensen MA., Fukushima M., and Davis RW. 2010. DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis. PloS
One, 5; e11024.
8. Sambrook J., Fritsch EF., and Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
9. KangJ., Lee MS, Gorenstein DG. 2005. The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J. Biochem Biophys Methods, 64; 147-151.
10.Musso, M., Bocciardi R., Parodi S., Ravazzolo R., and Ceccherini I. 2006.
Betaine, dimethyl sulfoxide, and 7-deazadGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn; 8; 544-550.
11.TG. Mammedov1, E. Pienaar1, SE. Whitney1, JR. TerMaat1, G. Carvill2, R.
Goliath2, A. Subramanian1, and HJ. Viljoen. 2008. A Fundamental Study of
the PCR Amplification of GC-Rich DNA Templates. Comput Biol Chem. 32: 52–457.
12.Skeiky YAW., Alderson MR., Ovendale PJ., Guderian JA., Brandt L., Dillon DC., Campos-Neto A., Orme IM., and Reed SG. 2004. Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as nacked DNA or recombinant protein. J. Immun.,172; 7618-7628.
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Published
2017-10-23
How to Cite
Sabrina, Y., Lestarini, I. A., Ekawanti, A., Sriasih, M., Depamede, S. N., & Ali, M. (2017). Improvements of PCR Amplification of Guanine plus Cytosine-Rich Constructs of Mycobacterium tuberculosis Gene using DMSO. Jurnal Kedokteran, 1(1). https://doi.org/10.29303/jku.v1i1.45
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