The ligase chain reaction in a PCR world.

S i n c e its discovery in 1985, the polymerase chain reaction (PCR) has had a profound impact on detecting genetic and infectious diseases, identifying new genes, and unraveling the mysteries of protein-ligand recognition.(1-s) Its universal utility is due to the exquisite specificity of amplification and ease of cycling made possible by the cloning and careful characterization of a thermostable polymerase from Thermus aquaticus. (6,7) Likewise, cloning of a thermostable ligase enabled a new amplification method, termed ligase chain reaction (LCR), to both amplify DNA and discriminate a single base mutation. (8-1~ Although these DNA amplification techniques are new, they bring to fruition the "enzymes as reagents" philosophy expounded by A. Kornberg and I.R. Lehman a quarter of a century ago. Allele-specific LCR employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand (see Fig. 1, top left). Thermostable DNA ligase will covalently link each set, provided that there is complete complementarity at the junc~ tion. (8) Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified and is therefore distinguished (Fig. 1, top right). A second set of mutantspecific oligonucleotides is used in a separate reaction to detect the mutant allele. This review will give a brief introduction to (1) determinants of specificity in amplification reactions; (2) differences between thermostable and thermophilic enzymes; (3) detection of single-base substitutions in DNA diagnostics; (4) characterization of DNA ligases; (5) cloning of DNA ligases; (6) use of ligases in DNA detection; (7) other methods that use ligase, such as ligase-mediated PCR and the branch capture reaction; and (8) potential new uses of ligase in PLCR and nested LCR amplification reactions. It will close with speculations on future prospects.