Detection and Identification of Mycobacterium Species Using Gene Amplification Techniques

Abstract

Gene amplification techniques or polymerase chain reactions (PCR) are laboratory procedures that use oligonucleotide primers to direct the amplification of a particular, hopefully diagnostically informative, target sequence to a detectable level (reviewed in Mullis and Faloona 1987). Basically, in these techniques, one begins by denaturing genomic DNA to produce single strands of DNA. Next, oligonucleotide primers are bound to complementary sequences on the target DNA. The primer-target DNA complexes are then elongated by DNA polymerase to produce two copies of the target. Importantly, DNA polymerase can only elongate or replicate DNA to which a primer is bound, so that the specificity of the amplification process is determined primarily by which primers are used. Finally, the sample is subjected to repeated cycles of denaturation, primer binding, and elongation to accomplish amplification of the target sequence. Since each cycle of amplification results in a twofold increase in the target sequence, this procedure is capable of amplifying a specific target sequence several-million-fold in about 25 cycles. Theoretically, such an amplification should allow the detection of a single target sequence in a sample.