1 材料与方法
1.1 DNA 样品
收集137份健康受试者外周血样品，用基因组DNA纯化试剂盒 (Promega公司)并按操作说明从全血中提取基因组DNA，紫外 (DU® 640, Beckman coulter) 定量后冻存于-20°C备用。
1.2 寡核苷酸合成
本实验采用的基因芯片检测CYP2C9基因多态性的方法参见文献[10]。寡核苷酸（引物或探针，序列见表1）采用标准亚磷酰胺化学方法在自动合成仪(ABI 8909，美国应用生物系统公司)上合成。所有荧光引物在合成中用Cy3亚磷酰化试剂在5’端进行荧光标记；所有分型探针在3'端进行氨基修饰。寡核苷酸合成完毕用浓氨水在55°C作用15h切割/脱保护，OPC柱纯化；用于纯化测序的引物以PAGE纯化。 Table1 Oligonucleotide used in this study | | Sequence (5'-3') | Application |
| F1 R1* F2 R2* | CAC ATG GCT GCC CAG TGT CAG CTT C GGC CAC CCC TGA AAT GTT TCC AAG ACG TGT GAT TGG CAG AAA CCG GAG C GGG ACT TCG AAA ACA TGG AGT TGC AG | Forward (F1, F2) and reverse (R1, R2) primers used to amplify the target fragments containing SNP sites. Reverse primers were fluoresceintly labeled at 5' end. |
| Mf1 Mr1 M2f Mr1 Mr2 Mr3 | ATT GAG GAC TGT GTT CAA GAG GAA GC CTT CCT CTT GAA CAC AGT CCT C CCT ACA CAG ATG CTG TGG TGC AC CTG GTG GGG AGA AGG TCA AGG TAT CTC CTG GTG GGG AGA AGG TCA GTG TAT CTC CTG GTG GGG AGA AGC TCA ATG TAT CTC | Primers used to construct mutant templates. The variant bases were introduced when synthesized. |
| P1 P2 P3 P4 P5 P6 P7 P8 | TTG AGG ACC GTG TTC AA - spacer- NH2 TTG AGG ACT GTG TTC AA - spacer- NH2 GAG ATA CAT TGA CCT TC - spacer- NH2 GAG ATA CCT TGA CCT TC - spacer- NH2 GAG ATA CAT TGA CCT TC - spacer- NH2 GAG ATA CAC TGA CCT TC - spacer- NH2 TAC ATT GAC CTT CTC C - spacer- NH2 TAC ATT GAG CTT CTC C - spacer- NH2 | Pairs of probes with one base difference (indicated with underline) for SNP discrimination, immobilized on aldehyde-coated slides surface with 3' end primary NH2 group. |
1.3 PCR 扩增
在一个PCR反应体系中采用F1/R1和F2/R2两对引物同时扩增两个目标片段。荧光引物不对称PCR反应条件（扩增产物用于杂交）：20 μl反应体系中含有200μmol/L dNTP, 0.1μmol/L 上游引物, 1μmol/L下游引物（Cy3荧光标记）, 40ng 基因组 DNA 或20ng 质粒DNA, 1×PCR 缓冲液和0.5 U Taq聚合酶。PCR扩增 (TECHGEN公司，USA)条件设置为：预变性 (94°C，5 min)；40 循环：变性 (94°C，30 s), 退火(58°C，30 s) 延伸(72°C，30 s)；延伸：72°C，5 min。
