Supplementary table1: Primers for microsatellite loci around pfdhfr

S. No. / Locus / Steps / Sequence / Cycling parametersa
1. / +0.3 Kb / Primary / GGAAATGCACATGTTTATAATAATC / 940C for 30 sec., 580C for 40 sec., 600C for 1:20 mins. for 40 cycles.
2. / AAATAATTTTAACAAATAGATATTCTTC
3. / Nested / GATTATAAGACAGATGTATGTGTAAC / 940C for 30 sec., 590C for 40 sec., 600C for 20 sec. for 35 cycles.
4. / CTTAAACAATCATATGATAAAAGGAC
5. / +1.28 Kb / Primary / AAAATGAAACAGTATGTAGTGCAC / 940C for 30 sec., 610C for 40 sec., 600C for 1:10 mins. for 40 cycles.
6. / ACTAGCTAGCATTAAAGAAGTTCC
7. / Nested / TTTGATTAGTTGGTTAATGTCGCC / 940C for 30 sec., 610C for 40 sec., 600C for 30 sec. for 35 cycles.
8. / ACAAAAACTAATACAAGAAGCTAAAG
9. / +5.6 Kb / Primary / GAGTTATATAATTTTTTTTATTATC / 940C for 30 sec., 570C for 40 sec., 600C for 30 sec. for 40 cycles.
10. / ATGAACAGGAAGTAATAATAAAC
11. / Nested / ATTAAAGGACAATATCTTTTTTATG / 940C for 30 sec., 570C for 40 sec., 600C for 30 sec. for 35 cycles.
12. / GGGAACATTTAATTAATTTCGG
13. / +9.8 Kb / Primary / TGGAGGGGATCAAAATAAAGAAC / 940C for 30 sec., 590C for 40 sec., 600C for 1.00 min. for 40 cycles.
14. / GAGCTTTTGATAAATAGTTTGTTTG
15. / Nested / ACATTTACCTTTTCAACTATTGATC / 940C for 30 sec., 590C for 40 sec., 600C for 30 sec. for 35 cycles.
16. / TATTTTTTCCCGTTTCATCCATAC
17. / +23.7 Kb / Primary / GCGTGTAGAAAACAAAATAATGCG / 940C for 30 sec., 630C for 40 sec., 600C for 1:50 mins. for 40 cycles.
18. / AGAATAATTTATGCACACCGCAAG
19. / Nested / GAATTTCTAGATTTATTTTCTTCG / 940C for 30 sec., 570C for 40 sec., 600C for 30 sec. for 35 cycles.
20. / CCATTGTTAGGATTCATTATTGG
21. / +28.5 Kb / Primary / GGCTAATTATGTTGACCGCATCC / 940C for 30 sec., 630C for 40 sec., 600C for 2.00 mins. for 40 cycles.
22. / ATCACAGTGGCTCTTGGTATACC
23. / Nested / GAAATATATAGATGTGTTTAGTAC / 940C for 30 sec., 550C for 40 sec., 600C for 30 sec. for 35 cycles.
24. / AATCATCACATTTATAATAAAGGC
25. / -0.3Kb / Primary / CATTCCATTTATATAAGTATGATTAG / 940C for 30 sec., 590C for 40 sec., 600C for 1:20 mins. for 40 cycles.
26. / CTTGTTCTTCCCATAACTACAAC
27. / Nested / ATCTGTTTAAAAGTTATATTAAGGGG / 940C for 30 sec., 600C for 40 sec., 600C for 30 sec. for 35 cycles.
28. / CATTCACATATGTTGTAACTGCAC
29. / -3.9 Kb / Primary / CTCACCCATCAGATATATCGTTG / 940C for 30 sec., 630C for 40 sec., 600C for 1:20 mins. for 40 cycles.
30. / ATATGGTAGTTATGTAGCGCAAAG
31. / Nested / TTCAATTATATTGCTGCAAAGG / 940C for 30 sec., 570C for 40 sec., 600C for 30 sec. for 35 cycles.
32. / GGAAATTATGTTATACAAAAGTG
33. / -5.2 Kb / Primary / ATTTGGAATTGCACGGAAGCAAC / 940C for 30 sec., 610C for 40 sec., 600C for 1:20 mins. for 40 cycles.
34. / GCAGGGAAATACATTTTGAGAGC
35. / Nested / CATATCAGATATGCGCACCTATG / 940C for 30 sec., 610C for 40 sec., 600C for 30 sec. for 35 cycles.
36. / ACAAATAGGTGTGTTCCCATAAAC
37. / -7.7 Kb / Primary / GGTTCATTGTATACTTTAAGCCTC / 940C for 30 sec., 610C for 40 sec., 600C for 1:50 mins. for 40 cycles.
38. / ACATACAAATGAAGGTCGATTTTAG
39. / Nested / CCAATGAGAAGAACAAAACAATAC / 940C for 30 sec., 590C for 40 sec., 600C for 30 sec. for 35 cycles.
40. / GAAAAAAACGGTTGATTCATTGTG
41. / -20 Kb / Primary / TGAGGAGAACGTACTGTCTTGC / 940C for 30 sec., 600C for 40 sec., 600C for 1:20 mins. for 40 cycles.
42. / GTTTGCCCTTGTTTAAAATGATTTG
43. / Nested / CGCTTTTAACATATTATTACCCATC / 940C for 30 sec., 600C for 40 sec., 600C for 30 sec. for 35 cycles.
44. / CTAAAATGCTTATCCCCTAGTTGC
45. / -30 Kb / Primary / GTATAATAACATTTTGCTTCAAC / 940C for 30 sec., 560C for 40 sec., 600C for 1:30 mins. for 40 cycles.
46. / TTCCTCATAACTATTCTTTTTAAC
47. / Nested / CCATAAATACATACACATGTGTAC / 940C for 30 sec., 590C for 40 sec., 600C for 30 sec. for 35 cycles.
48. / CTATAAAGCCACCTGGATAAGG

a Initial denaturation of 5 min at 940C and final extension of 7 min at 600C were used for all set of primers.

Supplementary Table 2: Frequency of pfdhfr alleles among Indian P.falciparum isolates of different geographical regions

pfdhfr allele / U.P.
(n=34) / A & N
(n=60) / Assam
(n=31) / Orrisa
(n=35) / M.P.
(n=30)
A16N51C59S108I164 / 0 / 0 / 0 / 0.4 / 0.2
A16N51C59N108I164 / 0.17 / 0 / 0 / 0 / 0.10
A16N51R59N108I164 / 0.83 / 0.13 / 0.81 / 0.54 / 0.37
A16I51R59N108I164 / 0 / 0.16 / 0 / 0.06 / 0
A16N51R59N108L164 / 0 / 0.03 / 0 / 0.33 / 0
A16I51R59N108L164 / 0 / 0.87 / 0 / 0 / 0

Note: - U.P.: Uttar Pradesh, M.P.: Madhya Pradesh, A & N: Andaman & Nicobar;

mutated amino acids are in bold faces