EVALUATION OF THE UTILIZATION OF REAL-TIME MELTING CURVE ANALYSES ON A 3D-POLYACRYLAMIDE DNA MICROCHIP FOR THE GENOTYPING OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE MDR1 GENE

Abstract

In this proof-of-principle study, I evaluated the utilization of real-time melting curve on re-usable 3D-polyacrylamide gel-based microchip (SNP chip) for SNP genotyping using SNPs 12/1236(T/C), 21/2677(G/T/A) and 26/3435(C/T) of the multidrug transporter MDR1. Using MCA approach, dissociation temperature (Td), the temperature at which 50% of starting duplex remained intact and the difference in Td between perfectly-match (PM) and 1-bp mismatched (MM) (I?Td) duplexes were calculated. Clear discrimination with I?Td of 7 a?? 11oC and 3 - 7oC was observed for synthetic targets and 90bp-PCR amplicons of SNP 26/3435(C/T), respectively. The sensitivity of SNP chip was evaluated using different C:T ratios (1:1 to 1:10). Heterozygous genotype cannot be distinguished when the C:T ratio is a?Y1:5. An in-house SNPgenotype program in MicrosoftA? Excel using Visual Basic for Application was developed to facilitate SNP genotype assignment for single SNP. In comparison to dideoxy sequencing, the genotypes of 58 out of 60 samples were correctly assigned for single SNP 26/3435(C/T), with 96.7% accuracy. Genotyping of multiple SNPs was evaluated using SNPs 12/1236(T/C), 21/2677(G/T/A) and SNP 26/3435(C/T) with a??wafflea?? SNP chip, consisting of smaller gel pads (10 x 10x 10A?m) covering an area of 300 x 300A?m, for better hybridizatrion and diffusion efficiency. For multiplex PCR, the PM and MM duplexes were successfully discriminated, enabling correct genotype assignment of SNP 12/1236(T/C) and 26/3435(C/T), with the exception of SNP 21/2677(G/T/A) due to weak hybridization signals on the corresponding mismatched probes.