【作者】 郭秋平；

【导师】 李庆阁；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 实时PCR是近几年发展起来的新型核酸检测技术，在临床诊断中具有极大的应用价值。与常规PCR检测技术相比，实时PCR集扩增检测于一体，大大提高了核酸检测的自动化程度，有效地消除了PCR产物污染。尤其是，实时PCR具有卓越的定量能力，灵敏度高，线性范围宽，结合各类探针技术，更具有极高的特异性。 该论文涉及一种新的实时PCR方法的建立和应用，论文分为四部分： 1．孪生引物实时PCR检测方法的建立。提出一种新型的实时PCR检测技术——孪生引物实时PCR。孪生引物是根据荧光淬灭和碱基配对原理设计的两条互补的DNA双链结构，其中一条DNA链是作为PCR扩增引物，称之为正引物；另一条与正引物序列完全互补，称之为负引物。我们以检测人β-珠蛋白基因为模型，建立了标记型和非标记型孪生引物实时PCR方法，证明该方法能抑制非特异扩增和防止引物二聚体生成。 2．孪生引物实时PCR基因分型技术。设计两种分别带不同荧光标记的孪生引物，采用实时PCR同时检测野生型和突变型模板，首次实现了非探针式单碱基突变的实时PCR基因分型。该技术速度快，易于自动化，特别适合大量单碱基突变基因的筛查。 3．孪生引物实时PCR检测端粒酶活性。端粒酶催化生成的核酸产物由许多短的重复序列组成，用一般的PCR方法检测容易形成引物二聚体，由于引物二聚体与特异扩增产物的序列相同，无法用基于探针或荧光染料的实时PCR特异检测。我们利用标记型孪生引物特有的特异扩增能力，实现了端粒酶活性的实时PCR检测。 4．用荧光淬火双链探针研究核酸酶的活性。在孪生引物的基础上设计出荧光淬火双链探针，建立了研究DNase Ⅰ酶切活性的实时 示踪方法。 另外，利用双激光荧光毛细管电泳技术研究了分子信标在实时 PCR中的作用机理，据此提出了采用轻微不对称PCR扩增能获得更 高的检测灵敏度，并用实验加以证实。更多还原

【Abstract】 Real-time PCR is a newly developed technique for nucleic acids detection, and has become increasingly important in clinical diagnostics. Compared with conventional PCR detection methods, real-time PCR combines amplification and detection in one step, and is easy to be automation. PCR product contamination is also prevented because of the close-tube detection format. Particularly, real-time PCR is good at gene quantification, not only with high sensitivity, wide dynamic range, but also with high specificity when introduced with a variety of probes.This dissertation is related with the development of a new type of real-time PCR and its applications, and contains the following four parts.1.The establishment of a "twin primer" real-time PCR. A new type of real-time PCR detection system-"Twin primer" real-time PCR was established. "Twin primer" has a double-stranded DNA structure and is labeled with a flurophore and a quencher. One strand of twin primer is used as extension primer named "positive primer". The other strand is complementary to the positive primer and is named ’~negative primer". Using ~ -globin gene as a model, both labeled and unlabeled types of twin primer real-time PCR were established. The results showed that twin primer real-time PCR could prevent non-specific amplification and primer-dimers.2.Twin primer real-time PCR genotyping method. Two kinds of twin primer, each labeled with a different flurophore, were used to discriminate wild-type and mutant type by real-time PCR. This is the first report of genotyping by non-probe real-time PCR. This method is characteristic of high speed, easy automation, and is especially suitable for high throughput screening.3.Detection of telomerase activity by twin primer real-time PCR. The catalyzed products of telomerase are made up of a series of repeated short sequences. It is difficult to detect telomerase with probe- or non-probe-based real-time PCR method. By using labeled twin primer PCR and its ability to prevent non-specific amplification, real-time PCR detection of telomerase were realized.4.Study on nuclease activity with fluorescence quenching probe. Based on twin primer design, we developed a general real-time method to trace nuclease activity.In addition, the mechanism of molecular beacon in real-time PCR was investigated by capillary electrophoresis with double-laser induced fluorescence detection. Based on the proposed mechanisms, a slight asymmetric PCR method was carried out to enhance the detection sensitivity. The validity of the method was proved by the experimental results更多还原