CYP450热点突变基因芯片分型方法的建立

Genotyping of hot-spot CYP450 SNPs with DNA microarray

  • 摘要: 目的: 探索适合多位点同步检测的SNP扩增方案和杂交检测条件,建立芯片杂交信号判读的标准。方法: 设计多重扩增的引物和探针;优化荧光标记引物的多重PCR条件。自行制备氨基化片基并点样制片,激光共聚焦扫描后判读检测结果。结果: 野生与突变探针杂交信号比值大于4或小于2.5对分型结果的判断有意义,而当信号值在2.5~4范围内应重新检测分析。结论: 通过多片段同步扩增和不同位点平衡杂交的实现,本方法适于流行病学调查,体现了寡核苷酸芯片对药物代谢酶多态性基因分型的优势。

     

    Abstract: Objective: To setup the protocol of multiplex PCR which suit the synchronizing hybridization and cutoff of signals discrimination.Methods: Optimisation of the primers and probes had been done;some key characteristics to PCR were adjusted and the final strategy was done.Preparing the oligoneucleid microarray,cutoff to determine wildtype and matant alleles was calculated.Results: It was recommended that ratios of wildtype allele to mutant signal which was higher than 4 or lower than 2.5 which be used as cutoffs in determination.When the ratio was in the range from 2.5 to 4,PCR or hybridization should be rerun.Conclusion: Multiplex PCR and synchronizing hybridization are helpful in epidermetology;the advantages of genechip is showed in CYP450s study by these techniques.

     

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