Oligonucleotide Ligation Assay (OLA)
The OLA consists of two phases, a multiplex PCR amplification and a multiplex OLA, in a single-tube format. In the first reaction a PCR primer is hybridized to the target sequence. The primers are designed with either the normal or mutant nucleotide(s) at the 3' end and a tail of different lengths to distinguish various PCR products based on size at the 5' end. A PCR reaction is performed.
The second reaction is a ligation reaction. A common primer contains a fluorescent dye marker (FAM, TET, HEX) at the 3' end and meets the first primer right over the nucleotide position that will be altered in a mutant allele. If the 3' end of the first primer matches perfectly with the target DNA, it will be brought into close enough proximity to the second oligonucleotide that both primers can be ligated together. No ligation occurs when there is a mismatch between the 3' end of the first primer and the target DNA.
View an animation of the Oligonucleotide Ligation Assay.
Advantages
Rapid, easy, single-tube analysis, high throughput capability on capillary sequencer, genotyping and automation capability.
Disadvantages
Requirement for an automated sequencer.
Example of a commercial kit
Cystic Fibrosis Genotyping Assay (Abbott Molecular
).
Last changed: 2010-01-25
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