Gov genotyping by PCR-SSP and PCR-RFLP analyses.

CD109 exon 19 (open box) and flanking introns are diagrammed. The Gov SNP (*) lies at the 5′ end of exon 19. Relative positions of the PCR primers (arrows) used to generate the 225 and 448 bp PCR products used for genomic SSP and RFLP analyses are shown. (A) Allele-specific antisense oligonucleotide primers differing by a single 3′ nucleotide corresponding to the Gov SNP yield allele-specific 225 bp bands. All reactions also contained control HGH primers that resulted in a 429 bp product (control). In addition, although the reactions shown in lanes 1, 3, and 5 containedGov^a-specific primers, those in lanes 2, 4, and 6 contained Gov^b-specific primers. (B) TheGov^a allele contains a single BstNI site (filled triangle) that is common to both alleles. The Gov SNP results in an additional BstNI site (open triangle) that is specific to the Gov^b allele. As a result,BstNI digestion of the 448 bp Gov^a-specific PCR product yields 2 fragments of 163 and 285 bp. In contrast, digestion of the Gov^b-specific product yields 3 fragments of 285, 81, and 82 bp. M, 100 bp DNA ladder; uncut, no BstNI added; aa/ab/bb, Gov genotype.