Genetic variation of TLR-4, TLR-9 and TIRAP genes in Iranian malaria patients

Malaria Journal

Table 1 Primers and profiles used for PCR-RFLP of the tlr-4, tlr-9 and tirap genes

			Temperature °C/time (min)
Genes	Primer	Sequence	A	E	D	C	Product size (bp)	Restriction Enzyme	Cut Product Size (bp)
	299TLR4F	ATACTTAGACTACTACCTCC ATG	56 (1)	72 (1)	94 (1)	35	213	*NcoI* (Fermentase)	G: 19 + 194
tlr-4*	299TLR4R	TTGTTGGAAGTGAAAGTAAG
	399TLR4F	TGTTATCAAAGTGATTTTGGGAG AA	54 (1)	65 (1)	94 (1)	35	185	*Hinf I* (Fermentase)	I: 162 + 23
	399TLR4R	AGGTAAATGAGGTTTCTGAGTGATAGG
	-1237TLR9F	TTCATTCAGCCTTCACTCAG							T: 264+145+126+23
*tlr-9*	-1237TLR9R	TCAAAGCCACAGTCCACAG	64 (1)	72 (1)	94 (1)	30	558	*BslI* (Fermentase)	C: 264+115+126+ 30+23
	-1486TLR9F	TTCATTCAGCCTTCACTCAG	64 (1)	72 (1)	94 (1)	30	558	*AflII* (Fermentase)	T: 413 + 145
	-1486TLR9R	TCAAAGCCACAGTCCACAG
	180 TIRAP F	AGTGCTGTACCATCGA CCTGCTG
tirap **	180 TIRAP R	TTCCCCTTCTCCCTCCTGTAGTAG	60 (1)	72 (1)	94 (1)	35	161	*Eam1105I* (Fermentase)	S: 141 + 20

All primers described in this study were designed in our laboratory (GenBank accession no. AF177765.1 for tlr-4; NW-001838877.2 for tlr-9 and NT-033899.7 for tirap) except 299TLR4F primer that was described previously [26]. The bold and underline nucleotides located in the 3' of forward primers indicate a mutation, and create a restriction site accordingly.
A: Annealing, E: Extension, D: Denaturation, C: No. of cycles
* tlr-4 (gat) D299G (ggt)
* tlr-4 (acc) T399I (atc)
** tirap (tcg) S180L (ttg)

ISSN: 1475-2875