Snapshot technique

Genomic DNA from EDTA-treated peripheral blood was extracted using the TIANamp Blood DNA Kit (Tiangen Biotech, Beijing, China). A total of 1797 individuals were genotyped for the G82S, -374T/A, and -429T/C polymorphisms located in the RAGE gene using the SNaPshot technique (Applied Biosystems, Foster City, CA, USA) as described previously [14 (link)]. The polymerase chain reaction (PCR) primers used for the G82S polymorphism were 5′-GCTGGGGTTGAAGGCTTTTTCT-3′ (forward primer) and 5′-CCGGACAGAAGCTTGGAAGGTC-3′ (reverse primer), and the primers used for the -374T/A and -429T/C polymorphisms were 5′-CCCATCTTGATTGCGCAAAGTT-3′ and 5′-TCAAAAAACATGAGAAACCCCAGAA-3′.

The genomic DNA was extracted using the phenol-chloroform-isopropyl alcohol method from 2 mL of blood collected in EDTA anti-coagulation tubes. The SNaPshot technique (Applied Biosystems, Foster City, CA, United States) was used to genotype SNPs. Polymer chain reaction (PCR) and extension primers were designed using the Primer5 software (version 5.00, PREMIER Biosoft International). The length of PCR fragments ranged from 80 to 240 bp. The PCR products were purified by phosphorylase (FastAP, Applied Biosystems) and exonuclease I (EXO I, Applied Biosystems) and subsequently extended using the ABI SNaPshot Multiplex kit (Applied Biosystems). The extension product was purified by FastAP and loaded on ABI3730xl (Applied Biosystems). The results were analyzed using GeneMapper 4.0 (Applied Biosystems). Primer sequences are listed in Supplementary Table 1. The SNPs in APOE (rs429358 and rs7412) were genotyped by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method as previously described (Corder et al., 1993 (link)).

The genomic DNA was extracted from blood (2 ml) stored in an ethylene diamine tetraacetie acid (EDTA) anticoagulation tube using the phenol-chloroform-isopropyl alcohol method. Polymerase chain reaction (PCR) and extension primers scheme were achieved through Primer 5 software (PREMIER Biosoft International, Version 5.00). PCR materials were conducted by purification with phosphorylase (FastAP, Applied Biosystems) and exonuclease I (EXO I, Applied Biosystems). A consequent extension was applied by the ABI SNaPshot Multiplex Kit (Applied Biosystems). Extended products were purified with FastAP and loaded into ABI3730xl (Applied Biosystems). GeneMapper 4.0 (Applied Biosystems) was used to conduct data analysis. The SNaPshot technique (Applied Biosystems) was utilized for genotyping of SNPs. The following SNPs were tested: rs10792832, rs11136000, rs11218343, rs1990620, rs1990622, rs3173615, rs34860942, rs3764650, rs3792646, rs3818361, rs3851179, rs4147929, rs56081887, rs5848, rs6656401, rs6701713, rs6733839, rs704180, rs744373, rs9331888, and rs9637454. The SNPs rs429358 and rs7412 of the APOE gene were determined by Sanger sequencing. Details of primers were described in Supplementary Table 1.