Verification Of Single Nucleotide Polymorphism Associated With Cancer Susceptibility And Application Of New Detection Method Based On Nano Material

Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by a single nucleotide mutation at the genomic level. SNP is considered to be a definitive factor which will decide the differences between human disease susceptibility and drug reaction. It can result in the susceptibility differences between different individuals on complicated diseases such as malignant tumor, etc.Polymerase chain reaction with confronting two-pair primer (PCR-CTPP) is a new technology to detect SNPs which is established on polymerase chain reaction with sequence specific primer (PCR-CTPP) and multiple PCR technology. This technology is helpful to select the susceptibility genes from clinical and epidemiological sample of tumor. It’s also a great help to the research of cancer pathogenesis and cancer risk evaluation caused by carcinogen exposure.Capillary electrophoresis (CE) is a new type of liquid separation technology based on traditional slab gel chromatography. It is a developed technology after the high performance liquid chromatography (HPLC). According to the variety of DNA fragment (ion surface, molecular shape, etc.) which will lead to different mobility time, SNP can be detected with the driving force of high voltage. With this technology, SNP mutation types and location can be detected directly. At present, the laser induced fluorescence (LIF) is one of the most widely used CE detection methods for its highest sensitivity.As the derivative of graphene, graphene oxide (GO) is easily water-soluble and has good bio-compatibility. Graphene oxide has a characterization of fluorescent quenching effect. Based on resonance energy transfer theory, it has been applied to the analyze the content of bio-molecules or inorganic molecules according to the’Fluorescence Off-Fluorescence On’process.In this thesis, we built close cooperation with Wuhan Union Hospital and applied different methods to verify the relationship between gene polymorphism and tumor susceptibility. Then, we established a method to separate and detect the particular oligonucleotides sequence which is based on a FITC labeled DNA probe combined with graphene oxide by using CE-LIF. The main contents and results are summarized as followed:The DNA repair gene XRCC1polymorphism was detected based on PCR-CTPP technology and its association with glioma susceptibility was also discussed. Two different pairs of primers were designed according to three different SNP sites of XRCC1gene such as Argl94Trp (rs1799782), Arg280His (rs25489) and Arg399Gln (rs25487). After a PCR amplification reaction of conventional gel electrophoresis, the SNP allele of XRCC1was analyzed. The primer3’end was regarded as the starting point of triggering extension for its high stability. Under a proper PCR condition, if any mismatch occurred, the amplification efficiency would be very low. However, the PCR amplification would be successful when the pairing process was correct. The case-control study showed that XRCC1codon194Trp and399Gln allele were the high risk pathogenic genes of glioma among Chinese population. It was proved that this method could be used to study the correlation between gene polymorphism and genetic susceptibility of tumor through the experiment. This study provided evidence of potential role for XRCC1polymorphism in the genetic predisposition to glioma among Chinese population. At the same time, the analysis of correlation of this DNA repair gene and glioma may provide a deeper insight into the study of the genetic and environmental factors to cancer risk.To explore the relationship between gene polymorphisms of C667T gene and the incidence of esophageal cancer, the Meta analysis method was applied. Among the gene polymorphism study of folate metabolic pathway, the MTHFR C667T gene was most concerned. Many previous studies showed that MTHFR of C677T gene was associated with the risk of esophageal cancer, but the conclusion was inconsistent. In our study, the original data of each research was quantitative synthesized based on Cochrane system evaluation software of RevMan4.3by using the Meta analysis method. It could help us to receive more objective and credible conclusion by expand the quantity of samples. Through the analysis and comparison of the distribution of case and control group of MTHFR C677T heterozygous genotype (CT), mutant homozygous genotype (TT) and wild homozygous (CC), we found that those who carrying the CT genotype (OR=1.37,95%CI (0.79,1.84)) and TT genotype (OR=1.72,95%CI (1.12,2.43)) had a significant increasing risk of esophageal cancer incidence (P<0.05). The result also supported the conclusion that MTHFR C677T gene played an important role in the pathogenesis of esophageal cancer. The study found that those who carrying MTHFR C677T CT gene and TT gene type in Chinese Han population had a relative higher risk than other ethnic population. At the same time, it was also proved MTHFR C677T TT genotype was a higher risk factor for esophageal cancer than CC genotype.Since the XRCC1gene was proved to be a high risk pathogenic genes of glioma among Chinese population, we took the primer sequence of XRCC1gene Arg194Trp as a target sequence and tried to build a new method basded on a FITC labelled DNA probe combined with graphene oxidetoto for capillary electrophoresis-laser induced fluorescence (CE-LIF) separation and detection of such particular oligonucleotides sequence. The FITC labeled DNA probe was absorb by graphene oxide and the fluorescence of FITC was quenched. Then the particular oligonucleotides T1in solution were recognized by FITC-DNA/GO complex. When the target sequence was existed, FITC-DNA was dissociated from the graphene oxide by the complementary base pairing between DNA bases and formed into FITC-DNA/T1. The fluorescence of FITC was recovered. Quantitative analysis of T1could be applied according to the "Fluorescence OFF-Fluorescence On" process. The results showed that by applying the technology of CE-LIF, it could be easily to separate the unreacted FITC-DNA/GO complex from FITC-DNA/T1. It has reached the purpose to dissociate before the fluorescence detection process and thereby greatly improving the detection sensitivity. When further improved, this strategy can be used as a detection method to quantitative target DNA. It is hoped be a new method to SNP research.