A rapid method to isolate (GT)n repeats from yeast artificial chromosomes.

Simple repeat polymorphisms, like (GT)n repeats are often multiallelic systems with high heterozygosity, which may substantially increase the number of informative families in linkage studies. Here I describe a rapid method for the isolation of (GT)n repeats from yeast artificial chromosomes (YACs), derived from approaches already described for the isolation of YAC ends (1), and for the isolation of simple repeats from DNA cloned in plasmid or phage vectors (2, 3). The YAC DNA is cut with a restriction enzyme, ligated to a linker containing a non-complementary region (Vectorette), and sequences flanking (GT)n repeats are amplified between a (GT)n primer and a primer corresponding to the region of non-complementarity of the Vectorette (Figure 1). To get rid of yeast DNA, which contains (GT)n repeats, YAC DNA is isolated by pulse-field gel electrophoresis on 1 % low-melting agarose, purified using the GeneClean method (Bio 101), and resuspended in 10 /J of sterile distilled water. The YAC DNA is then digested in a 20 /tl volume with 1 unit of a restriction enzyme, to obtain fragments that can be ligated to the Vectorette. Hinfl was utilized for the experiment (details available on request). The reaction is stopped by heating at 65°C, and DNA is ethanol precipitated together with 0.6 pmoles Vectorette and 200 ng tRNA as carrier. The pellet is resuspended in 8 /tl sterile distilled water, then 1 /tl lOxligase buffer and 1 y\ T4 DNA figase are added. Ligation is performed for 1 hour at room temperature. Amplification is carried out with a modified (GT)n primer and the universal Vectorette primer (5'-CGAATCGTAACCGTTCGTACGAGAATCGCT-3'). The modified (GT)n primer (5'-G4(GT)M-3') has four additional Gs at the 5' end to avoid a smearing artefact that can be observed with a simple (GT)n primer even without template, possibly due to the formation of large multimers of the primer. Linkers can be added at the 5' end of the (GT)n primer as well (2), but they are not necessary in the method described here that takes advantage of the TA cloning system (Invitrogen Corporation, San Diego). Amplification is performed in a 100 y\ volume, with 0.5 fi\ template, 1.5 mmol/1 MgCl2, 2 mmol/1 of each dNTP, and 50 picomoles of each primer. Temperature cycles are: 5 minutes at 96°, 1 minute at 80° during which Taq polymerase is added, then a first cycle of 1 minute at 94°, 1 minute at 62°, 1 minute at 72°, followed by 34 cycles of 1 minute at 94°, 45 seconds at 69°, 30 seconds at 72°. When 10 /J of the reaction are analyzed on a 2 % agarose gel a set of discrete bands of the size expected for the restriction enzyme used in the Vectorette library construction is found, and no bands are detectable in a control reaction without template. Amplification products can be rapidly cloned by ligating 1 /il of the reaction with 50 ng of the PCR3000 vector, according to the TA cloning method. 20-30 white colonies, enough to be expected to contain all amplified fragments, can be rapidly screened by PCR, using a few bacteria as template and the primers described above. Inserts can be labeled and hybridized to Southern blots to prove their human origin, and for physical mapping. Inserts may be sequenced with the PCR3000, or with the Vectorette sequencing primer (5'-CGCTGTCCTCTCCTT-3'). An oligonucleotide primer is then constructed from the sequence of the CA strand of each clone of interest and the repeat itself, together with a stretch of flanking sequence on its opposite side, are amplified using the same Vectorette library, or another one from the same YAC clone.