PCR assay for GSTT1 and GSTT2B deletion genotyping.

A four primer set was used in a single reaction to determine the presence or absence of the GSTT1 allele. (A); Primers A-L and A-R amplify a 459 bp sequence present in the GSTT1 gene, while primers O-L and O-R amplify a joint sequence of 1460 bp resulting from the deletion of GSTT1. When GSTT1 is not deleted, the DNA fragment (GSTT1) between the forward (O-L) and reverse primers (O-R) is too long for amplification and only the 459 bp sequence is amplified. Conversely, when GSTT1 is deleted, the 459 bp sequence is not amplified because absent while the 1460 bp fragment is now amplified. As a result of the PCR reaction, the only presence of a 459 bp band (lines 1–3 in the gel picture) or of a 1460 bp band (lines 7, 8) defines the WT/WT and the del/del genotypes respectively; while the presence of both fragments defines the heterozygous genotype (lines 4–6). Lane 9 was used as negative control. Solid lines represent genomic sequences; white rectangle represents the deleted sequence; small gray triangles indicate the 408 bp repeats flanking the GSTT1 gene. Expected PCR products are drawn as small gray bars. (B); A three primer set is used to determine the deletion polymorphism of GSTT2B in a single reaction. Primers 2B and 6857 amplify a 847 bp fragment detecting the presence of the GSTT2B gene, while primers 6857 and 6858 amplify a 505 bp sequence resulting from the deletion of GSTT2B. The 6857 primer is used for amplification of both fragments. When GSTT2B is not deleted, the DNA fragment (GSTT2B) between the 6857 and 6858 primers is too long for amplification and only the 847 bp fragment is amplified. Conversely, when the gene is deleted, the 505 bp fragment is amplified and the 847 bp fragment is not amplified. As a result of the PCR reaction we can have three possible polymorphisms: one single 847 bp (lines 1–3 in the gel picture) or one single 505 bp band (lines 6–8) represent the WT/WT and the del/del polymorphisms rispectively; while the contemporary presence of both bands represents the WT/del polymorphism (lines 4, 5). Lane 9 was used as a negative control. Solid lines represent genomic sequences; black rectangle represents the deleted sequence. Expected PCR products are drawn as small gray bars.