Ligase chain reaction (LCR)--overview and applications.

1Department of Food Science, Cornell University, Ithaca, New York 14853; 2Department of Plant Pathology, New York State Agricultural Experiment Station, Corneli University, Geneva, New York 14456; 3Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, New York 10021 PCR has facilitated the development of a variety of nucleic acid-based detection systems for genetic disorders as well as for bacterial, viral, and other pathogens. (1) In the last few years, a number of other DNA amplification methods, including self-sustained sequence replication (3SR), (2) Q-beta replicase (QI3), (3) and the ligase chain reaction (LCR), (4's) have been developed to complement, or as alternatives to, PCR. (6'7) From its initial detailed reports in 1991, LCR evolved as a very promising diagnostic technique that is often utilized in conjunction with a primary PCR amplification. LCR employs a thermostable ligase and allows the discrimination of DNA sequences differing in only a single base pair (see Fig. 1). (4's) The power of LCR is its compatibility with other replication-based amplification methods. By combining LCR with a primary amplification, one effectively lines up the crosshairs to distinguish single base-pair changes with pinpoint accuracy. The intellectual genesis of LCR can be traced back to pioneering work by Whiteley et al. (8~ who described an oligonucleotide probe-based assay using two probes that are ligated together only when immediately adjacent to each other. The same concept is applied in the oligonucleotide ligation assay (OLA). (9'10) This method was used in conjunction with a primary PCR step to screen for sickle cell anemia, the AF508 mutation in cystic fibrosis, and T-cellreceptor polymorphisms. Wu and Wallace ~11t described a similar technique called the ligase amplification reaction (LAR), which employs two sets of complementary primers and repeated cycles of denaturation (at 100~ and ligation (at 30~ using the mesophilic T4 DNA ligase. Use of mesophilic, that is, T4 or Escherichia coli, ligase has the drawback of requiring the addition of fresh ligase after each denaturation step, as well as appearance of targetindependent ligation products. (11,~2) In contrast, LCR provides a much higher sensitivity and is less susceptible to the formation of false-positive ligation products. Thermostable ligase minimizes target-independent ligation because the reaction can be performed at or near the melting temperature (T,n) of the oligonucleotides. (s) Furthermore, the use of thermostable ligase avoids the need to add fresh ligase after each denaturation step as required in LAR. Recently, thermostable ligase has become available from a variety of commercial suppliers, and this will probably lead to even wider application and use of this new amplification technique. The concept of LCR and ligation-based diagnostics has been reviewed. (s'13) We will provide an overview of the recent advancements, new developments, and applications of LCR and similar ligase-mediated detection methods.