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Ligation Chain Reaction

Ligase chain reaction (LCR) is a method for amplifying DNA that uses a thermostable ligase instead of polymerase like PCR. Each cycle of LCR doubles the target sequence as the ligase joins two probes that hybridize to the target. LCR has greater specificity than PCR since it requires an exact match for the ligase to join the probes. LCR has been used to detect single nucleotide polymorphisms and genetic diseases.

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60 views

Ligation Chain Reaction

Ligase chain reaction (LCR) is a method for amplifying DNA that uses a thermostable ligase instead of polymerase like PCR. Each cycle of LCR doubles the target sequence as the ligase joins two probes that hybridize to the target. LCR has greater specificity than PCR since it requires an exact match for the ligase to join the probes. LCR has been used to detect single nucleotide polymorphisms and genetic diseases.

Uploaded by

amaya rajiv
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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LIGATION CHAIN REACTION

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 which thermostable
ligase is used to join two probes or other molecules together and amplified by standard
polymerase chain reaction (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.
LCR requires two completely different enzymes to operate properly: ligase, to join probe
molecules together, and a thermostable polymerase (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. 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.

Target conditions

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

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. Among these methods, LCR is emerging as the most sensitive method with high
specificity for known single-nucleotide polymorphism (SNP) detection. 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.

Ligase Chain Reaction Process

Appropriate DNA probe will selected to bind with target sequence. For instance, if your
target sequence is ATGC, then you will need a DNA probe with the complementary base
pairs of TACG. The DNA probe will ensure the target sequence is amplified. The LCR
process is carried out in three basic steps in a thermal cycler.

1. Denaturation - DNA is heated so that the double-stranded structure becomes single-


stranded. The hydrogen bonds are broken, allowing the base pairs to be exposed so
that the DNA probes can attach.
2. Annealing - Two sets of DNA probes attach to each end of the single strands of
DNA. This step marks where the duplication of the DNA will begin. From this point,
Taq polymerase will add nucleotides based on the already existing DNA strands.
3. Ligation – The DNA ligase will fill in the gap in the DNA strand between the probes
by filling in the appropriate primer. Ligase will only recognize and ligate primers that
properly anneal to the probes.
4. Separation – Gel electrophoresis is used for the separation of the amplified LCR
products. The target molecule is separated using PAGE.
5. Detection – Autoradiography is used to detect the LCR products. It is a technique
where the probes are labelled with radioactive molecules, which on exposure to X-
rays to be visualized.

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