Abstract

Genotyping N-acetyltransferase 2 (NAT2) for acetylation status of its enzyme is very important for personalized medicine, especially individualized dosing of anti-tuberculosis drugs and in bladder cancer epidemiology. Human NAT2 gene with at least 359 single nucleotide polymorphism (SNP) accessions is highly polymorphic which makes it difficult to design specific primers for allele specific PCR. Because of this, sequencing is the preferred choice to genotype for NAT2. Currently, a common tag SNP (rs1495741) and 7 SNPs (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208 and rs1799931) are found to be highly relevant with slow acetylation phenotypes. One of the latest contributions to related work is genotyping NAT2 with only two SNPs (rs1041983 and rs1801280) that outperforms the tagging SNP and is equivalent to the conventional 7-SNP NAT2 genotyping strategy. The aim of our work is to decrease the number of samples to be sequenced for one of the aforementioned SNPs (rs1801280 341T>C) using high-resolution melting analysis sequence matching function. It enables a prior elimination of the samples to be sequenced that are above a user-defined confidence threshold when genotypes are auto-called in comparison with sequencing and KASP confirmed reference samples. The workflow in screening type 1 SNP (341T>C) was shown with various remarks to experimental flow, such as improving the usability by gradient facility of thermal cyclers, HRM availability of Rotor-Gene 6000™ real-time PCR instrument and its software with auto-calling genotypes function, the commercially available EvaGreen™ based HRM kits and challenges of method.

Key words: NAT2, SNP, rs1801280, 341T>C, High-resolution melting, sequence matching, 5 × HOT FIREPol® EvaGreen® HRM Mix, SsoFast^TM EvaGreen™ Supermix, Type-it® HRM™ PCR Mix, Rotor-Gene 6000™.