Novel NAT2 haplotyping using allele-specific sequencing.

BACKGROUND

NAT2 is a common metabolizer of many clinical drugs. NAT2 haplotyping requires a complex procedure. Allele-specific PCR followed by direct sequencing or cloning sequencing are common methods used for haplotyping. However, these common methods require labor-intensive procedures. Allele-specific sequencing was designed for haplotyping of the NAT2 gene.

MATERIALS & METHODS: Using rapid DNA polymerase with high fidelity, we amplified the NAT2 coding region of genomic DNA for direct sequencing, allele-specific sequencing and for the cloning of genomic DNA from 307 healthy Korean subjects. Direct sequencing analysis of the 870-bp coding region of NAT2 was performed in order to search 11 of the most common SNPs. For cases who were heterozygous for two or more SNPs and whose haplotypes were not determined by direct sequencing, we performed sequencing analysis using the allele-specific sequencing primer for one specified allele. We performed cloning-sequencing analysis for confirmation of the haplotyping results of allele-specific sequencing.

RESULTS

Homozygotes for SNPs, heterozygotes for one SNP and heterozygotes for two or more SNPs were 142 (46.3%), six (2.0%) and 259 (51.8%) cases, respectively. There was 100% concordance between the results of NAT2 haplotyping using allele-specific sequencing and cloning sequencing of 65 cases that were heterozygous for two or more SNPs in 307 samples. For cases that were homozygous for the SNPs by direct sequencing, the haplotypes of NAT2 were clearly determined by cloning sequencing.

CONCLUSION

We have developed a novel method for NAT2 haplotyping using allele-specific sequencing, which could be an innovative and reliable method for NAT2 haplotyping.