Flanking region sequences and internal repeat structure of the pYNH24 (D2S44) 2 kbp insert analyzed by polymerase chain reaction and partial digestion with RsaI

The 2 kbp YNH24 pUC18 insert was partially sequenced, amplified with fluorescence-labeled primers, and characterized by incomplete digestion with the restriction enzyme RsaI. A characteristic RsaI profile with one restriction site in each repeat unit was obtained when the fragments were analyzed on a DNA sequencer. We developed this procedure with the specific aim of analyzing the internal repeat structure in recombinant alleles often observed in the YNH24 variable number of tandem repeat system (D2S44). Nonetheless, it should also be possible to apply the method when analyzing other tandem repeats, even when little information regarding their sequences is available. In addition, when the repeat array is amplified, either a 5' or a 3' fluorescence-labeled primer can be used to enable analysis from both directions of fairly long (> 3.6 kbp) alleles. The RsaI fragments obtained by partial digestion of the amplified pYNH24 insert corresponded well to the RsaI sites found in the sequenced regions. The 5' flanking region contained eight 30-34 bp sequences similar to the repeat unit and also several RsaI sites, whereas only a few RsaI sites and no repeat units were found in the 3' flanking region. Twenty-five consecutive RsaI sites were revealed, in the center of the pYNH24 insert, indicating a region with strictly repeated core sequences; this repetitive block comprised less than half of the total insert. Two approximately 2.24 kbp HinfI alleles from two different heterozygous individuals were found to have identical internal structure, which was very similar to that of the insert although it exhibited only 15 RsaI repeats.