Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR

International Journal of Systematic Bacteriology
B B StoneW G Weisburg

Abstract

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70 degrees C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete ...Continue Reading

Citations

Mar 6, 2008·International Journal of Systematic and Evolutionary Microbiology·Ho-Suk MunBum-Joon Kim
Jun 30, 2019·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·Mehdi Fatahi-Bafghi
Jul 15, 2005·International Journal of Systematic and Evolutionary Microbiology·Hong KimBum-Joon Kim
Mar 3, 2007·International Journal of Systematic and Evolutionary Microbiology·Ho-Suk MunBum-Joon Kim

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