Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism

Cytochrome P4501A1 (CYP1A1) is a member of the cytochrome P450 gene family and plays an important role in the metabolism of exogenous and endogenous material. In recent research, it has been shown that its genetic polymorphisms are associated with many diseases. But the isoschizomers such as the BsrDI enzyme required for the detection of this polymorphism are expensive.

The study used an improved PCR-RFLP method with mismatched base for detection of the single-nucleotide polymorphism rs1048943. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme StyI for RFLP was cheaper than other enzymes. A total of 320 samples from Han Chinese were tested to evaluate this novel method. The PCR results were confirmed by DNA sequencing.

After detecting 320 Chinese Han individuals, the genotype frequencies were 63.74% for AA, 31.54% for AG, and 4.72% for GG. The allelic frequencies were 75.48% for A and 24.52% for G. The x² test showed the genotype and allele frequencies of CYP1A1 do not deviate from Hardy-Weinberg equilibrium, and the sequences of amplified products were consistent with the one published in GenBank with the exception of mismatched base.

Based on PCR with mismatched primers we designed, the CYP1A1 polymorphism could be identified effectively with low cost.