DETECTION OF POINT MUTATIONS WITH A MODIFIED LIGASE CHAIN-REACTION (GAP-LCR)

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we e xplored the feasibility and limitations of a modified ligase chain rea ction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the lig ation of two pairs of synthetic oligonucleotides which hybridize at ad jacent positions to complementary strands of a target DNA, Multiple ro unds of denaturation, annealing and ligation with a thermostable ligas e result in the exponential amplification of the target DNA. A modific ation of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common pro bes plus an allele-specific probe were used, A single base mismatch ei ther at the ultimate 3' end or penultimate 3' end of the allele specif ic probe was sufficient for discrimination, though better discriminati on was obtained with a mismatch at the penultimate 3' position, Compar ison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and liga se on specificity, As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mu tations that confers AZT resistance, Mutant DNA could be detected and discriminated in the presence of up to 10 000-fold excess of wild-type DNA.