Oligonucleotide Ligation Assay (OLA)

The oligonucleotide ligation assay (OLA) provides another method for detecting well-defined alleles that differ by a single base. OLA uses a pair of oligonucleotide probes (oligomers) that hybridize to adjacent segments of DNA including the variable base. The oligomer on the 5' or left-hand side of the pair is an allele-specific oligonucleotide (ASO) to one allele of the target. The last base at the 3' end of this ASO is positioned at the site of the target DNA's polymorphism; the ASO also has a biotin molecule at its 5' end that functions as a chemical hook. The oligomer on the 3' or right-hand side of the pair is the common oligomer (sequence is the same for the two different alleles.) The common oligomer is positioned at an invariable site next to the target DNA's polymorphism and is fluorescently labeled at its 3' end.

If the ASO is perfectly complementary to the target sequence:

The ASO hybridizes completely when annealed and will lie flat against that target.
DNA ligase can then covalently join the ASO to the common oligomer.
The biotin hook is used to remove the ASO and the fluorescently labeled common oligomer will also be removed, producing detectable fluorescence.

If the ASO is not a perfect match to the target sequence:

The hybridization will not be complete. The 3' base of the oligomer will not be base-paired to the target DNA.
No ligation is possible.
When the biotin hook is used to remove the ASO, the common oligonucleotide will not be removed and therefore there is no detectable fluorescence in the molecule removed.

Testing to determine which allele is present:
To distinguish between two known alleles that differ by a single base, three oligonucleotides are necessary: Two are allele-specific oligonucleotides (ASOs) that differ from each other only in the single 3' terminal base; the first is complementary to one allele and the second is complementary to the second allele. The third oligonucleotide is complementary to the invariable sequence adjacent to the variant base.

Advantages of the ligase-mediated genotype over the PCR/hybridization protocol:

The chances of a false positive is virtually zero.
Because there is not electrophoresis, no filter hybridization, no washing of blots, the OLA protocol is much easier to automate and quicker to carry out.

Limitations:

OLA only works for single-base substitutions

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