Oligonucleotide ligation assay (OLA) is a highly specific method for detecting well-defined alleles that differ by a single base.24,25 The target sequence is initially amplified using PCR and then denatured. A pair of allele-specific oligonucleotide (ASO) probes (one specific for the wildtype allele and the other specific for the mutant allele), a common reporter probe (complementary to a sequence common to both alleles), and DNA ligase are added to the denatured PCR products. The ASO probes are designed to differ from one another only at the terminal 3' base. The common reporter probe is positioned immediately adjacent to the 3' terminal end of the ASO probes. If the ASO is complementary to the amplicon, DNA ligase can cova-lently join the ASO and the reporter probe. If the ASO is not a perfect match to the amplicon, the 3' base does not anneal with the amplicon, and DNA ligase cannot join the ASO and reporter probes. The ligation products are analyzed by electrophoresis. Alternatively, one of the probes can be biotinylated at the 5' end and the other probe tagged at the 3' end with a reporter molecule such as fluorescein or digoxigenin. If ligation occurs, the ligation product is biotinylated at one end, facilitating capture onto a strepta-vidin-coated microtiter plate. The opposite end contains the reporter label. Washing removes unbound label and the reporter molecule is detected.
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