The ligase chain reaction (LCR) is a method of

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

amplification. The ligase chain reaction (LCR) is an amplification process that differs from

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...

(PCR) in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard PCR cycling. 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, to join probe molecules together, and a thermostable

polymerase In biochemistry, a polymerase is an enzyme (Enzyme Commission number, 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 ...

(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 mutation A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequences ...

s; a single base mismatch at the junction of the two probe molecules is all that is needed to prevent ligation. By performing the ligation right at the T_m 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 certain

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 chromosome abnormality. Although polygenic disorders are ...

s. LCR and PCR may be used to detect

gonorrhea Gonorrhoea or 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. Gonorrhea is spread through sexual c ...

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 w ...

, 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 and bioinformatics, 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 ...

(SNP) detection (20). LCR was first developed in 1989 by multiple groups, 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