Analysis of Triplet Repeats of the FRAXA Locus Using a Novel Sequencing Procedure

Extract

Recent reports have shown that in normal alleles, the CGG repeat at the FRAXA locus is interrupted by one or two AGG and that abnormal alleles seem to be generated by expansion of pure CGG repeats at the 3′ end [1].

It is therefore important to establish rapid, simple and low-cost sequencing procedures for determining not only the number of CGG repeats but also the presence of AGG triplets.

A one-lane sequencing procedure with PCR-amplified DNA, labelled at the 3′ or 5′ end with a single fluorochrome, has been recently developed in our laboratory [2, 3]. This methodology is particularly suitable for detecting mutations in family studies [4] and has been applied to the analysis of the sequence of triplet repeats at the FMR1 gene after PCR amplification, using one of the two primers fluorescently labelled at the 5′ end.

The sequence of the primers utilised in the PCR reaction is reported by Erster et al. [5]. The direct primer was fluorescently labelled at the 5′ end with 5-(6)-carboxyfluorescein (Fluoreprime, Pharmacia) and synthesised using an ABI 391 PCR-Mate-EP DNA synthesiser. The PCR amplification was performed on 0,5 ug DNA in a 50-μ1 PCR buffer containing 10% DMSO, 10% glycerol, 25 pmol of the two primers, 0,3 mM dNTPs and 1 U Taq DNA polymerase.

Each sample was submitted to amplification on a Perkin Elmer 9600 PCR reactor, with the following cycling profile: 5 min at 95 °C, 30 cycles of 30 s at 97 °C, 1 min at 55 °C and 1 min at 72 °C, followed by a 10-min terminal extension at 72 °C. After precipitation with 5 M ammonium acetate, the pellet was dissolved in 80% (vol/vol) formamide and aliquots were heated for 2 min at 90 °C or, alternatively, for sequence analysis, for 10 min at 110 °C. The samples were loaded on 6 or 8% PAGE sequencing gels and analysed using an ABI 373A automatic sequencer as previously described [2].