A QUANTITATIVE ASSAY FOR ASSESSING ALLELIC PROPORTIONS BY ITERATIVE GAP LIGATION

A variety of techniques are currently available for detecting point mu tations in DNA, These techniques are frequently not sensitive enough t o be applied as quantitative assays in evaluation of relative occurren ce of alleles in cases of polymorphism or when variations in allelic g ene expression are being evaluated at the level of RNA, We report here the establishment of an iterative gap ligation (IGL) assay that is bo th quantitative and sensitive, The design of the assay is such that li gation of an upstream to a downstream primer across a single nucleotid e gap will only occur if the gap is filled with a deoxynucleotide comp lementary to the wild-type or mutant sequence, Under conditions in whi ch excess upstream primer saturates the template concurrently with lim iting amounts of downstream primer quantitative ligation is absolutely dependent on provision of the appropriate gap filling nucleotide. Whe n gap ligation occurs in a single incubation, or cycle, the amount of ligated product is a linear function of the relative amount of mutant sequence, with a sensitivity and detection limit of similar to 3% over a range of relative concentrations of 0-100%, When the reaction occur s over multiple cycles, or iterations, gap ligation becomes a non-line ar function such that small changes in the relative proportions of all eles produce a disproportionately large amount of ligation, As a conse quence, the sensitivity and limits of detection of the assay improve t o 0.2% after only 8 cycles, The development of this assay provides a u nique means of quantifying allelic polymorphisms in both DNA and RNA ( after initial amplification by PCR or RT-PCR) and should be applicable to any experimental settings in which nucleic acids from tissues or m ixed populations of cells are being evaluated.