Automated DNA Sequencing Analysis of MDR1

A total of 52 samples (19 fresh tumor and 33 FFPE) of ovarian tumor obtained from different patients were subjected for genotyping exon 12 (C1236T), 21 (G2677T/A) and 26 (C3435T) of the ABCB1 gene respectively by automated DNA sequencing (Applied Biosystems 3500 XL Genetic Analyzer). In order to compare the exon sequence data between cancer and non-cancer individual, the DNA isolated from the blood of 19 healthy individual were also used to genotype the above mentioned exons. The cycle sequencing-PCR reaction was performed following the manufacture’s protocol (Big Dye terminator reaction Kit version 3.1 Applied Biosystems, United States). The sequencing primers for genotyping of exon 12 (C1236T), 21 (G2677T/A) and 26 (C3435T) of MDR1 were designed manually and also verified by using Primer3 software¹. The list of internal primers used for cycle sequencing is shown in Table 3. The generated chromatogram of each of the exon sequenced was evaluated for the quality of sequence data by matching with standard reported sequence with the corresponding peak and SNPs were identified by analyzing the heterozygous or homozygous peak manually as shown in Supplementary Figure P2. The SNPs were further re-confirmed by comparing the heterozygous or homozygous peak in the tested DNA samples and control DNA by using nucleotide sequence analysis tools software (Finch TV). The identified SNPs were also re-confirmed by reverse strand sequencing.

Showing internal primers used in the cycle sequencing reaction for Automated DNA sequencing of exon of the MDR1 genes.