Genotyping And Rare Mutation Detection With Real-time PCR

Genetic polymorphism is an usual phenomena in human population. Thepolymorphisms may lead to human disease in certain conditions. The availability of rapid, specific molecular analysis to detect the various mutations has become increasingly important for prevention and control of disease. Somatic mutations, which often found in cancer, are good biomarkers for cancer detection and prognosis, or good targets of anti-cancer or relation marks with drug response. But somatic mutations are often rare mutations with a large amount of wild DNA background. So it's not easy to detect these somatic mutations. This dissertation describes real-time PCR with different solutions (displacing probes, ARMS primers, PAP primers and HRM) to test polymorphisms. This dissertation comprises 4 chapters. The former three chapters focus on genotyping, the fourth chapter describes detection of somatic mutations.Chapter 1 Genotyping of human platelet antigensIn chapter one, a four-color real-time PCR method using displacing probes was adopted as a solution for 6 HPAs genotyping. Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect the single nucleotide polymorphism responsible for each of HPA-1,-2, -3, -4, -5 and -15 genotype and with two HPA systems analyzed in a single PCR within 100min. After validation with samples of known genotypes, a total of 150 blood samples of healthy donors were genotyped. The genotyping results were compared with PCR-SSP, PCR-RFLP and sequencing, and the gene frequencies of each HPA were calculated. The validation results were in concord with their known HPA genotypes and all the 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP or sequencing. The gene frequencies of HPA-1 to -5, and -15 among the Chinese population in Xiamen obtained were consistent with previous reports of Chinese living in other territories.Chapter 2 Genotyping of ABO blood groupIn chapter two, a four-color real-time PCR was designed to genotype major ABO alleles. Seven displacing probes labeled with different fluorophors were used to detect O¹(261delG), A(261G), A(796C/803C), B(796A/803C),O²(802G>A), A²(1059delC), and A² (1009A>G), respectively. These probes were combined in one or two PCRs to determine major ABO alleles, single-tube format could reveal totally 6 genotypes corresponding to the 4 serotypes, whereas dual-tube format could reveal totally 15 genotypes. The single-tube PCR format could detect genomic DNA quantitatively ranging from 0.16 ng to 500 ng per reaction.110 human blood samples of known phenotypes were tested by single-tube or dual-tube PCR format and PCR-SSP method. Genotype analysis of 110 whole blood samples gave good concordant results with PCR-SSP method and also agreed with their corresponding phenotypes except one sample of O^I O^Iv-B genotype.134 DNA samples were tested in blind assay using single-tube PCR format. Only one result was disagreed with the record, but it was valid by sequencing.Chapter 3 HLA-B27 detecting by real-time PCRIn chapter three, a three-color real-time PCR was designed to detect HLA-B27 which is strongly associated with the disease ankylosing spondylitis. After aligning exon2 and exon3 sequences between HLA-B^*27 and other HLA-B alleles, a pair ofprimers and two displacing probes were designed, which could detect all 35 alleles of HLA-B^*27. The assay also contains an exogenous control labeled with HEX for the presence of inhibitors, which may lead to false negative results. A total of 362 clinical samples were tested and compared with flow cytometer method. The result of 359 samples were good concordant, but 3 samples gave disagreed results, the agreement was 99%.164 samples were positive and 198 samples were negative by using flow cytometer test; 165 samples were positive and 197 samples were negative by using real-time PCR method.Chapter 4 Detection of rare Gefitinib-sensitizing mutations of EGFR gene in NSCLCIn chapter four, 4 strategies were established for detecting rare Gefitinib-sensitizing mutations of EGFR gene in NSCLC and compared with each other. The four strategies were ARMS primer, dual-strand PAP primer, HRM and displacing probe, respectively. By using ARMS primer method, dilution for sensitivity studies revealed that hot-site mutations and other mutations were detectable in the presence of at least 0.1% and 1% EGFR mutation DNA, respectively; By using dual-strand PAP primer method, 0.01% 19-M1 under wild type DNA background could be detected clearly, for other hot-site mutations, 0.1% could be detected; By using HRM method, 20 of 29 mutations could be differentiated with wild type, and abilities of selectivity were about 1-10% for various mutations; By using displacing probe method, the ability of selective was 1% for 19-M1 and 5-10% for other deletional mutaions. The four methods were compared each other according to ability of detection, cost, popularity of instrument and the clinical situation, the ARMS method was considered as the best approach.12 samples were tested by ARMS method. 2 tissue samples with L858R were clearly detected by ARMS method and sequencing;1 pleural effusion with deletion mutation was only detected by ARMS method.