Ligase chain reaction
The ligase chain reaction (LCR) is a method of DNA amplification. While the better-known PCR carries out the amplification by polymerizing nucleotides, LCR instead amplifies the nucleic acid used as the probe. For each of the two DNA strands, two partial probes are ligated to form the actual one; thus, LCR uses two enzymes: a DNA polymerase (used for initial template amplification and then inactivated) and a thermostable DNA ligase.[1] Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR.[2]
Target conditions
It has been widely used for the detection of single base mutations, as in genetic diseases.[3]
LCR and PCR may be used to detect gonorrhea and chlamydia, and may be performed on first-catch urine samples, providing easy collection and a large yield of organisms. Endogenous inhibitors limit the sensitivity, but if this effect could be eliminated, LCR and PCR would have clinical advantages over any other methods of diagnosing gonorrhea and chlamydia.[4] Among these methods, LCR is emerging as the most sensitive method with high specificity for known SNP detection (20). LCR was first developed by Barany, who used thermostable DNA ligase to discriminate between normal and mutant DNA and to amplify the allele-specific product. A mismatch at the 3′ end of the discriminating primer prevents the DNA ligase from joining the two fragments together. By using both strands of genomic DNA as targets for oligonucleotide hybridization, the products generated from two sets of adjacent oligonucleotide primers, complementary to each target strand in one round of ligation, can become the targets for the next round. The amount of the products can thus be increased exponentially by repeated thermal cycling.
References
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- ↑ Wiedmann M, Wilson WJ, Czajka J, Luo J, Barany F, Batt CA. "Ligase chain reaction (LCR)--overview and applications." PCR Methods and Applications 1994 Feb;3(4):S51-64 PMID 8173509
- ↑ Barany F. "Genetic disease detection and DNA amplification using cloned thermostable ligase." Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):189-93. PMID 1986365
- ↑ New diagnostic tests for gonorrhea and chlamydia Gregory J. Locksmith MD. Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida, USA. Available online 13 February 1998.
General reading
- Walker, J. M., & Rapley, R. (2005). Medical biomethods handbook. Totowa, N.J.: Humana Press. ISBN 978-1-59259-870-0
