A New Type Of Real-Time Fluorescence PCR Detection System And Its ApplicationsSystem

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...