Genotyping of SLC variants
The genomic DNA was extracted from 3-4 ml of blood using the
phenol-chloroform extraction technique. The genotype of variations was
determined using the PCR-PFLP method as previously
mentioned.10,19,20 For amplifying a DNA fragment for
the SLC19A1 (G80A , rs1051266) variant
following sets of primers were used: forward primer 5’-
AGTGTCACCTTCGTCCCCTC-3’ and reverse primer 5’- CTCCCGCGTGAAGTTCTT-3′.
For SLCO1B1 A388G in exon 4 (rs2306283), the
set of primers used was forward primer 5’-ATAATGGTGCAAATAAAGGGG-3′and
reverse primer 5’-ACTATCTCAGGTGATGCTCTA-3’ and for SLCO1B1
T521C (rs4149056) variant, following sets of primers
were used: forward primer 5’-TTGTCAAAGTTTGCAAAGTG-3’ and reverse primer
5’-GAAGCATATTACCCATGAGC-3’. For amplifying a DNA fragment for theSLCO1B3 (A1683-5676G , rs11045585)
variant following sets of primers were used: forward primer
5’-GTGGGTAAAAGGCAGGTAAATG-3’ and reverse primer 5’-GAATTCAAACATCTC
ACTGTGCTC-3’. The PCR mixture of 20 µl comprised of 1X PCR buffer, 0.5
µM of forward and reverse primer, 1.5 mM MgCl2, 100
µg/ml bovine serum albumin (BSA), 200 µM dNTPs, 1U Taq polymerase
(DNAzyme, Thermo Scientific), and 200 ng of DNA. PCR conditions used for
the mixture were: 95˚C for 5 min and 94˚C for 30s (denaturation), 59˚C
(G80A /rs1051266), 55 ˚C
(A388G /rs2306283), 60˚C
(T521C /rs4149056) and 57 ˚C
(A1683-5676G /rs11045585) for 45s (annealing)
followed by 72˚C for 30s (extension) for the 29 cycles as well as the
final extension for 5 min at 72˚C. For SLC19A1
G80A , a PCR product of 230 base pairs (bp) was
digested with 5U of HhaI restriction enzyme (New England Biolabs)
at 37˚C. The wild-type alleles were recognized as GG (125, 68, 37 bp),
variant alleles were identified as AA (162,68 bp), and heterozygous
alleles produced both bands (162,125,68,37 bp)
(Figure2a ).10 ForA388G , the PCR product of 214 bp was checked on
2.0% agarose gel and then digested with 5U of TaqI restriction
enzyme (New England Biolabs), respectively at 65˚C. The wild type allele
(AA ) yielded bands of 151, 63 bp variant allele (GG ), were
identified by 128,63,23 bp, and heterozygous displayed all the bands(Figure-2b) . For T521C , a PCR product
of 209 bp was digested with 5U of HhaI restriction enzyme (New
England Biolabs) at 37˚C. The wild-type alleles were recognized as TT
(209 bp), and variant alleles were identified as CC (189, 20 bp)
(Figure2c ).19 ForA1683-5676G , a PCR product of 286 bp was
digested with 5U of Rsa I restriction enzyme (New England
Biolabs) at 37˚C. The wild type allele (AA) yielded bands of 262, 24 bp,
variant allele (GG) were identified by 155, 107, 24 bp, and heterozygous
displayed all the bands (Figure-2d) .20Digested fragments were separated on 8 % Native- polyacrylamide gel
electrophoresis (PAGE) and were stained with the ethidium bromide and
visualized under UV trans-illuminator. Two separate people looked into
the data to eliminate any potential biases, and 20% of the samples were
chosen at random, ensuring that the outcomes were 100% reproducible.