低拷贝DNA模板检验方法探讨

Study on the amplifying and typing of low copy number DNA

目的探讨扩增及检测方法对低拷贝DNA模板分型检测灵敏度的影响。方法Control DNA 9947A按比例稀释,采用IdentifilerTM和DNATyper15TM试剂扩增,循环参数设置为28和28+6个循环,平行扩增3次,分别单独检测及3次合并检测,使用310及3130分析仪检测。结果28+6个循环的基因座检出率高于28个循环;等位基因不平衡及丢失与基因座没有特异性的关联,随着DNA模板量的减少,等位基因不平衡及丢失增多;将3次扩增产物混合后检测,等位基因不平衡及丢失情况减少,分型正确率增高。结论模板DNA分3次扩增后混合检测、循环数为28+6,可提高低拷贝模板基因座的检出率。

Objective To study the influence on STR typing for low copy number （LCN） DNA using different methods of amplification and detection. Methods Control DNA 9947A was diluted and then amplified with Identifiler^TM or DNATyper15^TM. The heat cycles were set to 28 or 28 add 6. Each template was amplified three times in parallel, and then the amplified products or the product mixture of three amplifictions were deteced with 310 or 3130 Genetic Analyzer. Results The success rate of STR typing with the method 28 ＋ 6 cycles was higher than that of 28 cycles. There are no correlation between the allele imbalance or allele dropout and STR locus. With the reduction in the amount of template DNA, allele imbalance and dropout gradually increased, and the allele dropout was more common than allele imbalance when the amount of template DNA was very small. When the product mixture of three amplifictions were deteced, the occurrence of allele imbalance and dropout reduce obviously. Conclusion The success rate of STR typing of LCN DNA can be obviously increased by detecting the product mixture of three amplifictions in parallel combined with the 28 ＋ 6 heat cycle condition.