IN-VITRO EXPANSION OF GGC-GCC REPEATS - IDENTIFICATION OF THE PREFERRED STRAND OF EXPANSION

The human fragile-X syndrome, a major cause of inherited mental retard ation, is associated with expansion of the trinucleotide repeat GGC:GC C. Repetitive sequences in DNA are subject to slippage during catalysi s by DNA polymerases. We characterized the extent of slippage of synth etic GGC:GCC repeats by various DNA polymerases: Tag DNA polymerase, K lenow fragment of DNA polymerase I, DNA Sequenase(R), DNA polymerase-a lpha and polymerase-beta, as well as HIV reverse transcriptase, All of these enzymes were found to expand GGC:GCC repeats, with the most ext ensive expansion exhibited by Taq DNA polymerase, Starting with a temp late and primer, each 15 nucleotides (nt) in length, the product of on e round of synthesis by Taq polymerase is as long as 250 nt, Sequence analysis of cloned DNA fragments expanded by Taq polymerase indicates that expansion involves multiple triplet additions and that it is asym metric. The asymmetric distribution of terminal nucleotides in the exp anded product is consistent with active expansion of the GCC strand an d passive additions onto the GGC strand. The preferential elongation a nd expansion of the GCC strand was confirmed in studies utilizing long er repeats within a single-stranded M-13 template.