Ligase Chain Reaction (LCR)
Ligase chain reaction (LCR) is the method which detects a specific nucleotide sequence with some similarities to polymerase chain reaction. It is similar to polymerase chain reaction (PCR) as it uses DNA polymerase and specific primers designed to bind to the target. However, it is unlike to PCR as the primers are not used to amplify the DNA sequence between the primers. In the presence of target sequences, both primers will hybridize in such a way that one primer will lie directly to the second primer of the pair. In the presence of DNA ligase, these two primers are joined to form a single strand and the target sequence will have been effectively ...view middle of the document...
However, unlike PCR, the primers are not used to amplify the DNA sequence between the primers. Instead, if the target sequence is present, both primers will hybridize so that one primer will lie directly adjacent to the second primer of the pair. In the presence of DNA ligase, these two primers are joined to form a single strand and the target sequence will have been effectively duplicated. In the presence of a mutation or if the target sequence is not present, the primers will not bind and ligation will not occur. As the cycle is repeated, only the target sequence (if it is present) is amplified.
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. Thermostable DNA ligase will covalently link each set, provided that there is complete complementarity at the junction. 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. A second set of mutant specific oligonucleotides is used in a separate reaction to detect the mutant allele.
Since 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. Its universal utility is due to the specificity of amplification and ease of cycling made possible by the cloning and careful characterization of a thermostable polymerase from Thermus aquaticus. 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. 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.
Principle of Ligase Chain Reaction
The principle of LCR is based in part on the ligation of two adjacent synthetic oligonucleotide primers, which uniquely hybridize to one strand of the target DNA. The junction of the two primers is usually positioned so that the nucleotide at the 3' end of the upstream primer coincides with a potential single base-pair difference in the targeted sequence. This single base-pair difference may define two different alleles, species, or other polymorphisms correlated to a given phenotype. If the target nucleotide at that site complements the nucleotide at the 3' end of the upstream primer, the two adjoining primers can be covalently joined by the ligase. The unique feature of LCR is a second pair of primers, almost entirely complementary to the first pair, that are designed with the nucleotide at the 3' end of the...