A6 - PCR: Amplification and identification of products

Abstract

The protocol described in this chapter has been used to detect Mycoplasma pneumoniae and M. genitalium in clinical specimens. The polymerase chain reaction (PCR) parameters to be optimized include the composition of the mixture, the volume used, and the amplification cycle. Standard PCR protocols can be used for the amplification of most DNA target sequences. The PCR-amplified fragment is visualized on an electrophoresis gel by ethidium bromide staining. The choice of gel, agarose or acrylamide, depends on the fragment size. Several DNA molecular weight markers suitable for approximate size analysis of PCR products are commercially available. The chapter illustrates concentration of different components, such as enzyme concentration and deoxynucleotide triphosphates. In routine assays, a direct blot of PCR products (dot or slot blot) may be advantageous. This procedure requires no electrophoresis and no DNA transfer, but the drawback is that the PCR product size cannot be visualized.