摘要
目的建立一种简便易行的MDM2基因单核苷酸多态性(SNP)309位点的检测方法。方法用DNA提取试剂盒提取外周血DNA,建立MDM2 SNP309位点荧光定量PCR熔解曲线检测法。应用煮沸法处理血样取得粗制DNA,行荧光定量PCR检测,建立位点特异引物法检测MDM2 SNP309位点基因型。比较静置不同时间、反复冻融等处理以及存在血液学指标异常的血样经煮沸法提取DNA对荧光定量PCR扩增反应的影响,通过观察PCR扩增情况,评估不同处理方法对PCR扩增反应的影响。结果荧光定量建立MDM2 SNP309位点PCR检测方法。煮沸法提取的DNA可用于普通PCR、荧光定量PCR扩增体系,且煮沸法处理血样具有较广的适用范围。结论采用MDM2基因SNP309位点特异性引物行荧光定量PCR检测法是一种简便易行的MDM2基因SNP309位点检测方法,该方法有较好的临床适用性。
Objective To establish a simple,convenient method for genotyping of MDM2 SNP309.Methods Using SYBR GREEN PCR with Tm-shift primers and genomic DNA purified with TIANamp DNA blood kit,we established a new method to detect genotyping of MDM2 SNP309.And we also genotyped MDM2 SNP309 by SYBR GREEN PCR with Tm-shift primers using whole blood DNA extracted by boiling method.Furthermore,genomic DNA were purified using boiling method from different blood samples kept at room temperature for different time or treated with multiple freezing and thawing as well as hematologic abnormal blood samples.Quantitative PCR were carried out to observe the amplification efficiency of the obtained DNA templates to assess the DNA extraction method.Results We established a novel,SYBR GREEN PCR method for MDM2 SNP309 genotyping.DNA purified by both methods mentioned before were suitable for MDM2 SNP309 SYBR GREEN PCR.DNA extracted using boiling method could be used for polymerase chain reaction even when the blood sample were disposed in different ways.Conclusion In the present research,we established methods for MDM2 SNP309 genotype.Boiling method for DNA extraction is proved to be a simpler and quicker way of processing whole-blood samples compared with the DNA templates,and is effective for both PCR-RFLP and SYBR GREEN PCR.
出处
《临床肿瘤学杂志》
CAS
2011年第7期577-581,共5页
Chinese Clinical Oncology
基金
国家自然科学基金资助项目(200803150007)
