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KMID : 0357319920270010035
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Journal of the Korean Society for Microbiology
1992 Volume.27 No. 1 p.35 ~ p.44
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Primer Directed Amplification of Mycobacterium tuberculosis DNA in Clinical Specimens I. Primers and Reaction Condicions
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김상재
박영길/조상현/심명섭
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Abstract
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Efficiency of the four different sizes of DNA fragments in multicopy IS6110 sequence and of two fragments in Pab gene were compared in detection of M. tuberculosis genomic DNA and 245bp of the former and 418bp of the latter gene were found most
useful.
The primers designed to target 245bp were much more sensitive I detection of template DNA than those targeting 418bp DNA.
Cetyltrimethylammonium bromide(CYAB) permittig stable annesling of primers at high temperature made kit possible to do annealing and extension at the same temperature so as to curtail 3-steps PCR to 2-steps which was more efficient in detecting
template
DNA. Furthermore CYAB was able to efficiently remove Taq polymerase inhibitory substaces present in sputum specimens.
Carry-over contamination of PCR products could be prevented by 8-methoxy-psolaren(MOP) monoadducts or crosslinks between bases of double stranded DNA upon UV-irradiatio or by uracil N-glycosylase(UNG) destruction of amplified DNA having uracil in
place
of thymine. However UNG treatment was preferable because 8-MOP was not easy to handle and known as carcinogen.
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