Development Of Real-time PCR Genotyping Based On Allele Specific Primer And Displacing Probe Methods

This dissertation consists of two part for two hard-core methods used in real-time PCR. The first part is the development of real-time PCR genotyping using allele-specific primers, the second part is the development of another real-time PCR genotyping method using displacing probes.In chapter one, we developed a four-step protocol for real-time PCR genotyping of ADH2 gene using ARMS primers. Serious primer-dimer made difficult determine of ADH2 genotypes using traditional 3-step PCR method. Based on the melting temperature difference between the primer dimer and the amplicon, a four-step PCR protocol that could eliminate primer dimmer interference was established, where the fluorescence was measured at the forth step at 79 oC. A total 150 human blood genomic DNA samples were tested to validate this method, and the results were compared with electrophoresis approach. The results showed that all the human genomic samples could be accurately genotyped by this real-time PCR method.In chapter two, another new real-time PCR genotyping method using phosphorothioate modified allele specific primers with proofreading DNA polymerase was developed. Normal ARMS primers display misincorporation when used for real-time PCR genotyping with EvaGreen. Primer stabilisation is a key requirement to enable the application of proofreading to allele specific primers genotyping. Introduction of one or more phosphorothioate linkages into the primer 3′terminus enhance the inhibition of exonuclease activity of T4 DNA polymerase. The allele specific primer with phosphorothioate modified 3′terminus in the last nucleotide got the best results of the inhibition of exonuclease activity. And the real-time PCR genotyping with proofreading DNA polymerase got similar results, the phosphorothioate modification of the last nucleotide of the 3′terminus of the allele specific primers coupled with proofreading DNA polymerase could remarkably reduce misincorporation and enhance the amplification fidelity of PCR. In chapter three, a real-time PCR genotyping method of aldehyde dehydrogenase 2 using displacing probes was established. We genotyped ALDH2 alleles using two displacing probes, one 3'-FAM-labeled probe complementary to the usual ALDH2*1 gene and the other 3'-ROX-labeled probe complementary to the atypical ALDH2*2 gene. Taking advantage of displacing probes, we have developed a rapid and accurate real-time PCR genotyping method for ALDH2. The total analysis can be finished within 2 h with 100% accuracy as validated with 136 human genomic DNA samples. The developed method could be used for routine testing or population screening for ALDH2 genotyping.In the last chapter, the real-time PCR with displacing probes for HLA-A locus genotyping was developed. We designed 18 specific and 2 control displacing probes in the exon 2 and exon 3 regions of HLA-A gene. Two control probes were labeled with HEX, the specific probes were labeled with CY5, FAM or ROX respectively. Three specific probes and one control probe labeled with different fluorescences were put in one tube, and all probes performed PCR simultaneously in total six tubes for a sample to get the genotype.