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Introduction
Principle
Phases
Ligation is regulated by 3 factors
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Polymerase Chain Reaction-Oligonucleotide Ligation Assay (PCR-OLA) is a method to diagnose hereditary diseases
caused by mutation not affecting restriction endonuclease sites.
This method combines Polymerase Chain Reaction with the Ligation Assay.
PCR-OLA distinguishes between the ligation and the absence of ligation of two oligonucleotides.
A single nucleotide mismatch (at the site of hybridized oligonucleotides) prevents ligation and hence we can
distinguish between the wild and mutant genotype.
PCR-OLA can be understood with the help of an example. PCR-OLA is a sensitive, rapid and highly specific
method.
Developed by Grunstein and Hogness in 1975.
Introduction
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• . Oligonucleotide ligation assay combined with polymerase chain reaction (PCR-OLA) is a technique which can be used for
the detection of characterized sequence variations.
• Commonly known as the OLA, the Oligonucleotide Ligation Assay is an analyte specific reagent that is centered around the
hybridization of a PCR primer with an exact match to a target sequence.
• The Oligonucleotide Ligation Assay is a genotypic assay for the detection of resistance-associated mutations to reverse
transcriptase and protease inhibitors in Human Immunodeficiency Virus type1 subtype B.
• A pair of nucleotides designed to anneal to adjacent sequences within a PCR product – If perfectly hybridized – Joined by
DNA ligase.
• Oligonucleotide complementary to normal and mutant sequences are differently labeled and the products are identified by a
computer software.
Introduction
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Principle
The target DNA sequence is PCR amplified prior to the ligation step of the annealed probes.
One of the detection probes consists of a sequence complementary to the target sequence.
The second detection probe is fully complementary to the target sequence and serves as a reporter.
Detection probes bind next to each other.
The product is captured by anti-TAG on the microsphere surface.
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Phases
• The OLA consists of Two phases,
1. A multiplex PCR amplification
2. A multiplex OLA, in a single tube format.
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A Multiplex PCR amplification
• In this reaction a PCR primer is hybridized to the target sequence. The
primers are designed with either the normal or mutant nucleotide at the 3’
end and a tail of different lengths to distinguish various PCR products
based on size at the 5’ end.
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A Multiplex OLA
• This reaction is a ligation reaction. A common primer contains a
fluorescent dye marker at the 3’ end and meets the first primer right over
the nucleotide position that will be altered in a mutant allele.
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Ligation is regulatedby 3 factors
• The specificity of ligation is regulated by 3 factors
1. The hybridization of oligonucleotide.
2. The need for these primers to directly adjacent to one another.
3. The requirement that the oligonucleotide have two bases
perfectly complementary.
