24 - Nucleic Acid Probes and the Polymerase Chain Reaction

Abstract

This chapter focuses on the applications and limitations of nucleic acid probe hybridization and polymerase chain reaction (PCR). PCR is a rapid procedure for amplification of specific DNA or RNA sequences in vitro. It is a highly sensitive and specific technique, with which minute quantities of target DNA, theoretically as little as a single molecule, can be amplified until a sufficient concentration of nucleic acid is obtained, which is greater than the threshold of the detection system being used. Contamination of samples by 'rogue' nucleic acid molecules is one of the main problems of PCR, particularly if it is being used for diagnostic purposes. Although the PCR itself is relatively simple, the acquisition of target molecules from samples can be time consuming and complicated. A nucleic acid probe is a sequence of single-stranded DNA or RNA that can hybridize specifically with its complementary sequence via base-pairing on the target nucleic acid molecule. Common applications of DNA/RNA probes include Southern (DNA) and northern (RNA) blotting, dot/slot blots (DNA/RNA), colony and plaque hybridization and in situ hybridization. Nucleic acid probes have been applied in diagnostic microbiology, in particular probes are being used for culture conformation as an alternative to conventional laboratory' techniques. DNA probes are also used to detect slow-growing organisms, such as Mycobacteria spp.