The ligase chain reaction (LCR) is a method of DNA amplification. The ligase chain reaction (LCR) is an amplification process that differs from PCR in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...

(PCR) cycling (Barany, 1991). Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR. Thus, LCR requires two completely different enzymes to operate properly:

ligase In biochemistry, a ligase is an enzyme that can catalyze the joining (ligation) of two large molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the larger molecules or the enzym ...

, to join probe molecules together, and a thermostable

polymerase A polymerase is an enzyme ( EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base- ...

(e.g., ''Taq'' polymerase) to amplify those molecules involved in successful ligation. The probes involved in the ligation are designed such that the 5′ end of one probe is directly adjacent to the 3′ end of the other probe, thereby providing the requisite 3′-OH and 5′-PO4 group substrates for the ligase. LCR was originally developed to detect point mutations; a single base mismatch at the junction of the two probe molecules is all that is need to prevent ligation. By performing the ligation right at the Tm of the oligonucleotide probe, only perfectly matched primer:template duplexes will be tolerated. LCR can also be used to amplify template molecules that have been successfully ligated for the purpose of assessing ligation efficiency and producing a large amount of product with even greater specificity than PCR. Thus, LCR is not necessarily an alternative, but rather a complement, to PCR.

Target conditions

It has been widely used for the detection of single base mutations, as in

genetic disease A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...

s. LCR and PCR may be used to detect

gonorrhea Gonorrhea, colloquially known as the clap, is a sexually transmitted infection (STI) caused by the bacterium '' Neisseria gonorrhoeae''. Infection may involve the genitals, mouth, or rectum. Infected men may experience pain or burning with ...

and

chlamydia Chlamydia, or more specifically a chlamydia infection, is a sexually transmitted infection caused by the bacterium ''Chlamydia trachomatis''. Most people who are infected have no symptoms. When symptoms do appear they may occur only several wee ...

, 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. Among these methods, LCR is emerging as the most sensitive method with high specificity for known

single-nucleotide polymorphism In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently lar ...

(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

General reading

*Walker, J. M., & Rapley, R. (2005). Medical biomethods handbook. Totowa, N.J.: Humana Press. {{DEFAULTSORT:Ligase Chain Reaction Biochemistry detection methods DNA profiling techniques Amplifiers Molecular biology techniques

Target conditions

See also

References

General reading