Single-track sequencing for genotyping of multiple SNPs in the N-acetyltransferase 1 (NAT1) gene

Abstract

Background: Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.

Results: A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.

Conclusions: The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1.

Figure 1

Genotyping of the coding region of NAT1 by T-track sequencing on Alf Express™. (A) Displayed area from T382 to T417 (B) Displayed area from T531 to T582 (C) Displayed area from T618 to T661, with genotypes 640T/T; (D) 640T/G; (E) 640G/G; (F) – Displayed area from T735 to T797, A752T and T777C alleles not found.

Figure 2

NAT1 variants in the coding region assessed by G-track sequencing on ABI310 PRISM. (A) Displayed area from G418 to G476, genotypes 445G/G and 459G/G; (B) 445G/A and 459G/A heterozygote; (C) Displayed area from G544 to G590, genotypes 560G/G; (D) 560G/A; (E) Displayed area from G598 to G655, genotypes 613A/A and 640T/T; (F) 613A/A and 640A/G.

Figure 3

NAT1 alleles in 3'-untranslated region assessed by single A-track sequencing on Alf Express™. Displayed area from A1038 to A1104. (A) NAT1*4/*4 – wild type; (B) 1088T/A and 1095C/A heterozygote; (C) 1088A/A and 1095A/A homozygote; (D) NAT1*4/*11 allele; (E) NAT1*11/*11 allele.

Figure 4

NAT1 alleles in 3'-untranslated region by single A-track sequencing on ABI310 PRISM. Displayed area from A1104 to A1048. (A) NAT1*4/*4 – wild type; (B) 1088T/A and 1095C/A heterozygote; (C) 1088A/A and 1095A/A homozygote; (D) 1088T/T homozygote and 1095C/A heterozygote; (E) NAT1*4/*11 allele.