(精选)Capillary Electrophoresis Instrumentation-Theory and Application教学课件.pptVIP

Advanced Topics in STR DNA Analysis;Outline for This Section;;DNA Analysis by RFLP ;I think I might have been stupid, in some respects, if it werent for my psychedelic experiences. Kary Mullis, Ph.D., Nobel Prize Laureate, Chemistry, 1993 ;The Process of DNA Typing via PCR;; ;The Issues;Why Use Capillary Electrophoresis for DNA Analysis?;Capillary Electrophoresis System;Process Involved in 310/3100 Analysis;Separation;Electrophoresis Theory “Ok here’s my recipe idea called the electric pickle. Attach the hot lead to a screw and shove it in. The neutral lead goes in the other end. Turn out the lights and plug it in It glows and sizzles. The juicy ones work best” /cook;DNA and Electrophoresis “From a practical point of view it is disappointing that electrophoresis cannot be used to fractionate or analyze DNA on the basis of size” Olivera, Biopolymers 1964, 2, 245;Separation Mechanism;Issues with CE separations;In the early 1990s the real question was how to transition from a gel to a capillary;So what are sieving buffers?;Entangled Polymer Solutions;C C*;R;Mesh size (concentration) and molecular weight (chain length) will affect resolution viscosity limits applicable chain length;low;500 V/cm;500 V/cm;The Keys;How to Improve Resolution?;How Are Separations Performed in a 310?;Synthesis Procedure for PDMA (Molecular Wt = 1Million amu);4% PDMA (100K), Taps buffer;Commercial POP-4;Separation Issues;Injection;DNA-;Capillary;Stacking Effects;Ion Mobility Effects The Injection of DNA by voltage is described by [DNAinj] = E(?r2)[DNAsam](?ep+ ?eof) However this equation assumes no interfering ions are present. Cl- ions and other interferents will compete with DNA {DNAinj} = [DNAinj]/[other ionsinj] Ions such as Cl- have a higher charge/mass ratio and ?ep is greater ;Effect of Formamide Conductance on DNA Concentration;DNA;;Comments on Sample Preparation;Injection Study;Effect of Formamide on Peak Resolution and Sensitivity (Ro