| The microchip electrophoresis, integrating multiple sample handing processes with the actual measurement step, are of considerable recent interest. Such devices are also referred to as " Lab-on-a-chip" devices, and the great advantages of the analytical microsystem include high speed, high performance, integration, versatility , miniaturization and the potential for high throughput. Recently, various DNA-based analysis has been realized on the microchip system such as the simple DNA fragment separation, triplet repeat DNA sizing, and analysis of PCR products, but the domain of DNA analysis can be broken down into more specific categories of nucleic acid, sequencing and genotyping. Of these, genotyping is most pertinent to clinical diagnostic and forensic analysis as it involves ascertaining differences in genetic content from one individual to another, and the inherited disease and genetic predisposition to disease can thus be diagnosed based on genotype analysis. Angiotensinogen (AGT) gene polymorphism has been reported to be associated with the risk of essential hypertension. Conventionally, detection of polymorphism or PCR products by gel electrophoresis is not easily scaled to accommodate miniaturization, automation, and a large number of samples. Alternative electrophoretic formats such as microfabricated devices may provide powerful platform for simple, fast and sensitive polymorphism analysis and gene diagnosis.Objectives : 1. To develop a microchip-based electrophoretic method for rapid analysis of the AGT gene (-6) A/G polymorphism in Dalian Hans population. 2. Sensitive detection of the PCR products of tuberculosis and hepatitis C virus gene from clinical specimens by microchip electrophoresis. 3. To compare the performance characteristics of the established gel electrophoresis method with the novel microchip-based techniques.Methods: 1. A total of 123 cases with essential hypertension and 103 control subjects in a Chinese population were enrolled in this study. Blood was drawn and DNA extracted. The (–6) A/G polymorphism in the core promoter of AGT gene was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay followed by a microfabricated electrophoretic device and slab-gel electrophoresis as well. 2. PCR products from 15 selected TB and 32 HCV specimens which submittes for TB and HCV detection were analyzed by microchip electrophoresis and the established clinical method.Results: 1. Microchip-based electrophoresis can separate PCR products of AGT gene within 250s and the relative standard deviation (RSD) of the migration time was 0.12% - 0.26% < 1% (n=6). It was approximately seven times quicker in microchip electrophoresis than gel electrophoresis and the reagent consumption was ten percent of gel electrophoresis. Its detection limit was 640 times more sensitive than gel electrophoresis.2. The A allele frequency was very high in both groups (control: 0.7038, hypertensive:0.7073), it was almost identical in the different groups(χ2=0.024, P>0.05), there was also no significant difference in frequencies of the allele of G(-6)A between Dalian and Tibetan population (P>0.05). The genotype and allele distribution of AGT( G(-6)A) gene in the controls and EH patients followed Hardy-Weinberg equilibrium law. There was no significant difference in genotype and allele frequencies in different genders , there was also no significant difference in distribution of G/A polymorphism in different age groups.3. Analysis time for detection of TB DNA by PCR-Hyricomb was 2-3 hour . While,Microchip electrophoresis provided identical results in 270 second per sample . Microchip electrophoresis resolved the 93 bp of HCV in 210 second,achieving 100% sensitivity and specificity compared with the established method .The detection limit of microchip was approximately 12800 times higher than PCR-Hyricomb.The detection limit of microchip was 0.03125×102 copies per lμl compared with The detection limit of 5×102 copies per lμl on real ti... |
PDF Full Text Request