Detection Of Nucleic Acids Using Loop Mediated Isothermal Amplification And Fluorescence Resonance Energy Transfer And Their Application In Sports Research

Nucleic acid is the carrier of biological genetic information,and its integrity is very important for the survival and normal reproduction of organisms.The analysis of single nucleotide polymorphisms(SNPs)and gene damage is of great significance in current biochemical research and clinical diagnostics.The development of simple,rapid,highly sensitive and specific method for nucleic acid detection has broad application prospects.In the dissertation we have developed new methods for detecting SNP and DNA oxidative damage with high sensitivity by using Loop-Mediated Isothermal Amplification(LAMP),Capillary Electrophoresis(CE),Fluorescence Resonance Energy Transfer(FRET)and other detection techniques.These methods have been applied in many sport research fields such as endurance assessment,prevention of sports hereditary diseases.The main contents are as follows:1.A new method for detecting athlete endurance outstanding genotype ACTN3 SNP was established,which was based on ligation-based loop-mediated isothermal amplification(LAMP).This method designed stem-loop probes which were complementary to the target DNA sequence respectively.The probe ligation was used to initiate loop-mediated isothermal amplification reaction.Double-stranded DNA was stained with SBYR Green I dye for real-time fluorescence quantitative detection.The high sensitivity of SNP typing of ACTN3 gene was achieved according to the difference of fluorescence signals(the time corresponding to the maximum slope in real-time fluorescence curve.The quantitative detection limit of the synthetic DNA was 100 aM and the method can accurately detect 0.1% mutation frequency.In addition,the method was applied to detection of the genomic DNA samples,the results were consistent with DNA resequencing results.2.Based on the principle of ligation-based loop-mediated isothermal amplification,the simultaneous detection of multiple SNP in one reaction system was detected by CE.During the reaction,the fluorescent group was labeled on primer,and the ligation probes with different lengths were designed for different SNP loci.The ligation reaction could accurately distinguish the base mismatch.The amplification products were digested by restriction endonuclease,then the fluorescent products were separated by capillary electrophoresis and the fluorescence signals were detected by laser induced fluorescence.Fragment analysis was performed,and four types of SNPs were distinguished according to fluorescent products of different lengths.The detection limit of the DNA was 2 aM.3.A novel fluorescence resonance energy transfer(FRET)method by using cationic conjugated polymer PFP was developed to detect DNA oxidative damage.DNA repair enzymes were used to treat oxidative damaged DNA samples to obtain 3'-5'-phosphate nucleotide gap.DNA polymerase was used to label fluorophores and PFP was added to obtain PFP-DNA complex,and the degree of oxidative damage of DNA was detected by fluorescence resonance energy transfer.The experimental results showed that the method had high sensitivity and specificity,and the detection limit of genomic DNA was 1 ng/?L.Compared with the existing methods,the method solved the shortcomings of tedious steps,low sensitivity and low selectivity in DNA damage detection,and had good application prospects in clinical medical detection.