Oligonucleotide Ligation Assay

Oligonucleotide primers that which have annealed to adjacent sites on single-stranded DNA, such that there is no gap between them, can be covalently joined together by DNA ligase.

Oligonuclotide Ligation Assay
Fig. 3. Multiplex ligation-dependent probe amplification.

This is the basis for the oligoligation assay (oligonucleotide ligation [OLA]). Successive cycles of denaturation, annealing, and ligation result in a linear amplification of the target sequence. In contrast, standard PCR amplification produces an exponential increase in the target sequence. The adjacent ends of the primers must be perfectly matched with the target sequence, otherwise ligation will not occur. Thus, a single nucleotide or a few adjacent nucleotides can be interrogated (62,63). Multiplexing allows a number of nucleotides and/or mutations to be detected in a single-reaction tube (64). Currently, one of the primers usually has a fluorescent label so that the product can conveniently be detected on a fluorescent DNA analyzer. Although OLA is not strictly a PCR-based method, a new method has been developed in which an initial OLA is followed by a PCR, called multiplex ligation-dependent probe amplification (MLPA). As described next, MLPA is ideally suited to dosage testing of multiexon genes. A similar technique, in which the initial OLA is replaced by a PCR (universal primer quantitative fluorescent multiplex [UPQFM]) has also been described (65).

5.1. MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION Multiplex ligation-dependent probe amplification is a hybrid technique that combines an OLA with a QF-PCR. It is ideally suited to the measurement of exon copy number in multiexon genes, but it could be used for the quantitative assessment of any locus (66).

PCR product

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