INTRODUCTION

The basic principles of nucleic acid amplification technologies (NAT) and definitions of the various techniques are covered in Nucleic Acid–Based Techniques—General〈1125〉. The current chapter covers major techniques that result in amplification of targeted nucleic acid sequences. The most common NAT assay is the polymerase chain reaction (PCR), which was first described by Kary Mullis. This procedure has been further refined to amplify a DNA fragment starting from RNA (reverse transcription-PCR, or RT-PCR). Initially, PCR was used in a qualitative manner to amplify and detect DNA molecules because its exquisite sensitivity paired with its high specificity made it a useful tool for the detection of nucleic acid targets. Since its inception, the number of PCR applications has expanded rapidly, and the technique, which now includes quantitative and multiplex assays, is currently used in almost every field of research and development in biology and medicine. In addition to the changes and improvements to the original design of the PCR procedure, alternatives to PCR are techniques used to amplify target nucleic acids to generate RNA instead of DNA amplicons. The most commonly used techniques are nucleic acid sequence–based amplification (NASBA) and the transcription-mediated amplification (TMA) which are described here in detail. In contrast to PCR, which relies on incubating the sample at three different temperatures, NASBA and TMA are based on isothermal conditions.