Association of SNPs in genes involved in folate metabolism with the risk of congenital heart disease

Benjing Wang, Minjuan Liu, Wenhua Yan, Jun Mao, Dong Jiang, Hong Li, and Ying Chen

J Matern Fetal Neonatal Med, 2013; 26(18): 1768–1777

! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2013.799648

(1) SNaPShot multiple PCR: 12 pairs of amplification primers (with patent) were synthesized by Shanghai Generay biotech Co., Ltd. The total volume is 10 mL including 10 buffer (MgCl2), MgCl2 (25 mM), dNTP ( 2 mM), Primer Mixture by mixing 12 pairs of primers and DNA template. Denaturation at 95 C, 4 min; Denaturation at 94 C, 30 s, Annealing at 66 C, 50 s, Reduce 0.5 C per cycle, Extension at 72 C, 50 s,total 11 cycles; Denaturation at 94 C, 30 s, Annealing at 61 C, 50 s, Extension at 72 C, 50 s, total 24 cycles; Extension at 72 C, 10 min; Stored at 4 C. (2) PCR product purification: The total volume is 6 mL including SAP (1 U/mL), ExonI (5 U/mL) and PCR product. Reaction at 37 C, 80 min, Terminated at 80 C, 20 min, Stored at 4 C. (3) Label extension PCR: 12 extension primers were synthesized by Shanghai Generay biotech Co., Ltd. The total volume of 6 mL including 5 seq Buffer, Extension-primer Mixture by mixing 12 primers and purification product. Denaturation at 96 C, 1 min; Denaturation at 96 C, 10 s, Annealing at 62 C, 5 s, Extension at 60 C, 30 s, total 28 cycles; Stored at 4 C....