Pcr-Oligonucleotide Ligation Assay from Dried Blood Spots (Technical Briefs‪)‬

PCR followed by oligonucleotide ligation assay (PCR-OLA) is a molecular method that can be used for the detection of nucleotide sequence variants. During the past 10 years it has been used successfully for numerous diagnostic applications (1-15). PCR-OLA is based on the enzymatic ligation of two oligonucleotides that anneal next to each other onto the PCR-amplified target DNA. Even a single-nucleotide mismatch between the oligonucleotides and the template precludes the ligation. Automated PCR-OLA on a microplate format in combination with ELISA detection technology is a powerful method for testing numerous samples (2). One bottleneck for the effective use of PCR-OLA in population screening programs has been the time-consuming and tedious preparation of DNA samples that is necessary to eliminate PCR inhibitors (16). Dried blood spot (DBS) specimens on filter blotters are widely used for collection, storage, and shipping of blood samples in screening programs for newborns (17), and they have been used for genotypic confirmation of positive screening tests (18). PCR inhibitors can be eliminated from DBS specimens by eluting the spots (19, 20), by fixing the spots to the disk with methanol (21, 22), or by extracting DNA from the disk (23, 24). To increase the throughput of PCR-OLA, we treated DBS specimens with methanol and used the methanol-fixed samples for the PCR-OLA analysis of the major mutation (AG[U.sub.Fin]) (25), which is responsible for Finnish-type aspartylglycosaminuria (AGU; McKusick 208400). We tested the methanol treatment on DBS specimens collected on five different types of glass fiber or filter paper blotters. The results show that PCR-OLA from methanol-treated DBSs enables rapid and reliable detection of genetic variances.