chapter  6
12 Pages

Amplification by DNA Synthesis (PCR)

Currently, PCR, the standard method of DNA amplification, is the oldest and most developed procedure. Several newer approaches for nucleic acid amplification have been developed, having some advantages over PCR for particular purposes — greater amplification per cycle (TAS, 3SR, Q-replicase), isothermal reaction (3SR), or coupled amplification-mutation detection (LAR, LCR, LAS) — and may eventually gain more widespread use after further development. Long before the advent of

in vitro

nucleic-acid amplification systems, the use of nucleic acid hybridization for amplification had long been a problem; the major impediment was the sensitivity of the detection method. Rapid, nonisotopic, nucleic acid-based systems could only be formulated for target sequences that were highly abundant. The detection of rare sequences required the use of long assays, highly radioactive probes, and large tissue samples, since the most sensitive hybridization probes — those continuously labeled with

P can detect a sequence only when at least 10

–10

target molecules are present in a sample. A major breakthrough was the development of PCR. With PCR, rare sequences in minute tissue samples could be amplified 10

-fold or more in several hours, enabling the use of nonisotopic detection systems and oligonucleotide probes for rapid hybridization-based immunoassays. Since the first PCR study appeared in 1985, a multitude of modifications to the basic protocol have appeared, extending the procedure to a diverse range of applications.