Detection Of SNP From A Single Cell By Microfluidic Chip And Study On Biomarkers Of Hereditary Susceptibility Of Styrene

The discovery and utilization of single nucleotide polymorphisms (SNP) is a cornerstone of modern molecμlar genetics. In a point mutation, a single nucleotide base is replaced by a diffenert single nucleotide base(transitions if purine to purine or pyrimidine to pyrimidine, A→G, orT→C; transversions if purine to pyrimidine or vice versa, G→C or A→T).With the complete sequencing of the human genome, large numbers of DNA sequence variants, mainly single nucleotide polymorphisms are revealed. A large number of SNPs have been found in the human genome and deposited to public databases. As the third generation of genetic markers, SNP has been used extentively in gene mapping, disease-correlativity analysis, popμlation genetics and drug research.The analysis of SNP in the human genome may offer the key to understand genetic differences between individuals and disease states , and eventually improve medical treatments by allowing the prediction of genetically related disease risk and intoxicant response. To meet these goals, major international collaborative efforts have been made to carry out large scale genomic studies that require the determination of hundreds of thousands of genotypes performed in many individuals.Most of current single nucleotide polymorphism genotyping methods are still too slow and expensive in large association studies with hundreds or more SNP in a large number of DNA samples. With the developing of biochemistry, good engineering solutions are needed to make high-throughput genotyping a reality. However, SNP genotyping technology is rapidly progressing methods. A number of high-throughput genotyping methods have been developed and are being readied for routine use. Microfluidic chip technology originated from analytical chemistry , adopts microfabrication technologies to make microchannels on a chip about several square centimeters. The technology can integrate the sample's injection, separation and detection into a single chip. The advantage of microfluidics is high efficiency and low consumption.With the progress of microelectronics and other microfabrication techniques, the technology of microfluidic chip developed rapidly recent years, and began to play more and more important roles in chemistry, biology and medical instruments.Single nucleotide polymorphism is an important type of biomarker of susceptibility which indicate the reaction ability to exogenous compound metabolism. Different morphous of metabolic enzyme has different response during the procedure of exogenous compounds exert toxic effect on organism and determines reaction ability of organism to chemical intoxicant. As a resμlt , it can be taken as an important screening index to occupational contraindication. In humans, styrene metabolism has been well characterized. Here we evaluate the influence of individual genetic polymorphisms of drug-metabolizing enzymes on urinary metabolites after occupational exposure to styrene.The study on single nucleotide polymorphism is vital in the future in occupational diseases prevention. It can discuss the mechanism of intoxicant action on the molecμlar level and accelerate assessing reaction nature of intoxicant.【Objectives】1. To study a method which can detect single nucleotide polymorphism from a single cell by microfluidic chip and analyze the factors that influence on the amplication resμlts.2. To evaluate the influence of individual genetic polymorphisms of styrene- metabolizing enzymes on urinary metabolites and search for the susceptible biomarkers of styrene occupational hazard on the base of molecμlar epidemiology. Offer scientific theoretical basis for screening occupational people and monitoring health of styrene exposed popμlation group.【Methods】1. Whole blood was obtained from healthy volunteers and drawn into EDTA-coated tubes. Isolate single cell from whole blood using a glass capillary. Single cell DNA templates were prepared with different methods. CYP2B6 genotype were determined by a nested polymerase chain reaction-restriction fragment length polymorphisms method(NPCR-RFLP)from a single cell. Then using micmfluidic chip technology and agarose gel electrophoresis to detect SNP, respectively.2. Occupational contact workers of styrene were included in the study. This study was conducted with the consent of all subjects. A questionnaire was administered concerning health status, smoking, alcohol consumption and medication. The subjects were divided into a low-exposure group(<100 mg/m3)and a high-exposure group(≥100 mg/m3)according to their styrene exposure levels. We applied the method of PCR-RFLP to determine the SNPs of CYP2B6, CYP2D6, GSTP1 and NAT2, and statistically analysed the influence of gene polymorphisms on the metabolism of styrene. The level of significance was set at 0.05. The statistical analysis was performed using SPSS software for Windows version 11.5.【Resμlts】1. Enzyme lysis method is the most efficient procedure for preparing the single cell DNA template,with a success rate (SR) of 96.7℅, while the SRs of alkali lysis and freezing-thaw lysis methods are 85.2℅ and 72.1℅,respectively. The nested polymerase chain reaction technique is efficient for single cell genotyping. The chip based method is found to be very sensitive,requiring much less sample and only quarter the time compare to agarose gel method.2. Detecting the genotype of NAT2, GSTP1, CYP2B6 and CYP2D6 of 58 workers exposed to styrene through collecting peripheral blood, we can find the level of urine styrene metabolites increment is influenced by genotypes of CYP2B6 and GSTP1. The metabolism of CYP2B6 G/G homozygotic genotype to styrene is more active than G/T heterozygotic genotype and T/T homozygotic genotype. The level of PHEMA in GSTP1 mutation genotype subjects is significantly higher than that in the group of homozygotic genotype.But the influence of NAT2 and CYP2D6 genotypes on urinary metabolites is not observed in the same study. Genotypes of CYP2B6 and GSTP1 are related to susceptibility to the metabolism of styrene in human.【Conclusions】1. The NPCR- micmfluidic chip technic is significantly more rapid, sensitive than agarose gel electrophoresis method for detection of SNP .2. Our resμlts suggest that although urinary styrene metabolites are good biomarkers of internal styrene dose in occupational exposure, genetic susceptibility of the individual shoμld also be considered in biological monitoring of exposure to styrene.