Development of New Analytical Buffer Systems for the Separation and Analysis of PCR Amplified DNA by Capillary Electrophoresis, Final Report

• NCJ Number: 198318
• Date Published: September 2002
• Length: 64 pages
• Grant Number(s): 1999-IJ-CX-K014
• Agencies: NIJ-Sponsored
• Publication Type: Report (Study/Research)

Annotation

This study focused on the development of methods to amplify and detect specific regions of the DNA molecule by using the polymerase chain reaction (PCR).

Abstract

The impact of the PCR has resulted in rapid and dramatic advances in biochemical analysis and has also resulted in a need for efficient and automated procedures for analysis of the reaction products. For many years it has been recognized that capillary electrophoresis (CE) has great potential for fulfilling this requirement. Capillary-based systems can produce rapid and efficient separations of DNA due to the efficient heat dissipation of the capillary when compared to standard slab gel methods. In an effort to develop a better understanding of the mechanisms for capillary failure, this study focused on two major issues: sample injection effects and band shifts. A series of experiments examined the role that temperature, ionic strength, buffer pH, and sample concentration played in DNA typing that used CE. The findings indicate that sample concentration and the choice of denaturing solvent can have a major effect on the quality of the output electropherogram. Band shifts clearly are the result of temperature effects; and the fact that a band shift can occur at one locus and not another is due to a differential response from locus to locus as temperature changes. The reason for these temperature effects is likely the secondary structure of the DNA, since changing to highly denaturing buffers at pH 11 greatly mitigates this effect. 14 figures and 21 references