Rapid PCR analysis of the St14 (DXS52) VNTR

Some segments of the human genome exhibit polymorphism due to a variable number of tandem repeats (VNTR). The enzymatic amplification of VNTR loci can allow for rapid analysis and discrimination of closely sized alleles. We report a PCR-based method for analysis of the highly polymorphic Stl4 VNTR. Source and Description: The S t l4 -1 probe is a 3 kb EcoRI fragment cloned from human DNA which detects several polymorphisms at the DXS52 loci, including a highly polymorphic VNTR RFLP revealed by Taql. Analysis of this VNTR by Southern transfer has revealed ten alleles ranging in size from 3.4—6.6 kb, with alleles as large as 15 kb in Black individuals (1, 3). The region responsible for the Taql polymorphism was sequenced from a cloned allele, and was found to be an almost perfectly duplicated 60 bp repeat (2). To assay the VNTR by PCR, amplification primers were designed on either side of the repeat. However, due to additional repetitive sequence flanking the hypervariable region, the PCR primers were not placed directly adjacent to the VNTR. As a result, amplified products include about 650 bp of flanking sequence. PCR Amplification: We used 1 /xM in each of the primers 5'GGCATGTCATCACTTCTCTCATGTT-3' and 5'-CACCACTGCCCTCACGTCACTT-3', 'standard' PCR buffer and dNTP concentrations (4), 1 /tg genomic DNA, and 24 cycles of PCR consisting of 20 sec at 94°C, 30 sec at 55°C, and 20 sec at 74°C, with a final incubation for 5 min at 74°C. Less favorable results were obtained with additional cycles. Size differences between the amplified products were found to correspond with those seen on Southern blots; Mendelian inheritance of the allelic products was also verified (data not shown). Frequency: 50 unrelated Caucasian males were analyzed using the above primers. The amplified allele sizes and frequencies observed are: 3000 bp (2%), 2900 (8%), 2400 (12%), 1690 (36%), 1630(2%), 1570(14%), 1390(10%), 1300(2%), 1220 (2%), and 700 (12%). We have also seen other rare allelic products (880, 1750, 1810 and 2100 bp) that were not present in these 50 samples. All of these products (except for die 880 bp product) are shown below. The size of the smallest product (700 bp) suggests that it represents an allele with just one 60 bp repeat; and the most frequent product of 1690 bp appears to contain 17 tandem repeats. Chromosomal Location: The DXS52 loci map to Xq26-28, and are linked to several disease genes including hemophilias A and B, fragile X syndrome, and adrenoleukodystrophy (2). The Stl4 VNTR specifically maps to Xq28 and is about 2 cM from the hemophilia A-coagulation factor VDI locus, yet its large number of alleles makes it very useful in the diagnosis of hemophilia A.