Variation in the gene coding for the M5 Muscarinic receptor (CHRM5) influences cigarette dose but is not associated with dependence to drugs of addiction: evidence from a prospective population based cohort study of young adults

Table 4 List of oligonucleotide primers, PCR conditions and restriction enzymes used to discriminate genotypes and allele frequency in Victorian European Caucasian population of putatively functional human muscarinic acetylcholine receptor M5 gene polymorphisms.

			PCR Conditions
SNP id	Primer Sequence Forward	Primer Sequence Reverse	Tm (°C)	Mg++ (mM)	Restriction Enzyme	Minor Allele Frequency
rs661968	gaagcattcaatgaatcttcaataagttct	atgcggtagatgaagactcc	55	2.5	MseI	C = 0.03
rs7162140	cattttgctgaccctaaagacc	tggacgcactacctttaaaaac	60	2	TaqI	T = 0.19
-257A>T	taacagcccctttgtgaacg	cctgtgggagaaatgtggtt	60	2.5	NlaIII	A = 0.03
rs2702309	ggcaaacaacttgacccacagaaaacga	cctgtgggagaaatgtggtt	60	2	MboII	A = 0.00
rs2702304	gaaaaaagtgactatgacaccccaaactag	tgattttcaccttgtgacagc	59	2	SpeI	T = 0.00
rs2576302	atggctgtcacaaggtgaaaat	tgccagtacaacttctcttcca	60 to 54 TD	2	DdeI	T = 0.00
rs2705353	atggctgtcacaaggtgaaaat	tgccagtacaacttctcttcca	60 to 54 TD	2	AlwNI	A = 0.00

Tm, annealing temperature for PCR amplification; TD, touchdown cycling conditions; Mg++, magnesium concentration for PCR amplification; touchdown, refers to -0.5°C change in Tm per amplification cycle.