利用TaqMan技术定量检测基因表达的引物探针设计  

Primer-probe design in quantitative analysis of gene expression using TaqMan technology

作　　者：张行[1] 叶景佳[2] 魏群 陈萍[1] 王琦[1] 曹江 

机构地区：[1]浙江大学邵逸夫临床医学研究所,浙江杭州310016 [2]浙江大学肿瘤研究所 [3]浙江省生物治疗重点实验室

出　　处：《全科医学临床与教育》2007年第6期468-471,F0002,共5页Clinical Education of General Practice

基　　金：浙江省科技厅项目(021103004)

摘　　要：目的建立优化的实时荧光定量PCR的引物探针设计参数。方法设计引物和探针时的原则包括:①上、下游引物的融解温度(Tm值)应尽量相同或相差越小越好,以利于PCR退火温度的优化;②扩增区域应当跨越不同的外显子(在基因组DNA上至少一个以上的内含子),以利于区分基因组DNA扩增产物和cDNA扩增产物;③引物和探针的位置最好位于外显子和内含子交界处,以避免引物和探针与基因组DNA的结合;④扩增区域最好靠近基因的3’端,以减少不完全逆转录的影响。本次研究设计了实时荧光定量RT-PCR检测人甲胎蛋白mRNA的引物和探针,并提取临床肝癌组织标本中的RNA,检测其中甲胎蛋白的表达。结果本次研究设计的实时荧光定量RT-PCR检测甲胎蛋白mRNA的引物和探针具有良好的特异性,可用于临床标本的检测。结论本次研究确定的原则对提高利用TaqMan技术定量检测目的基因结果的可靠性具有很好的指导意义。Objective To set appropriate parameters for primer - probe design in quantitative analysis of gene expression using TaqMan technology. Methods Some general principals for the design of primer pairs and probes： ①the calculated melting points （Tins） of beth primers should be the same or with the least difference for easy optimization of the annealing temperature in PCR ; ②the amplicon should cover different exons （with at least one large intron in genomic DNA） for easy discrimination of improper amplification of genomic fragments from cDNAs ; ③it would better that primers and/or probes be located at the boundaries of exon - intron to avoid misannealing of primers or probes to genomic DNA templates ; and ④the amplicon should be near 3＇ end of the gene to minimize the loss of templates due to incomplete reverse transcription. The design for primer pair and probe for detecting human alpha fetoprotein mRNA was illustrated in detail in this paper. Results The primer - probe set designed showed high specificity in detection of human fetoprotein mRNA in clinical samples. Conclusion The principles established in this study are very useful in providing reliable results of TaqMan technology.

关 键 词：TAQMAN技术 基因表达 定量PCR 

分 类 号：R450[医药卫生—治疗学]