Sizing and quantitation of polymerase chain reaction products by capillary electrophoresis for use in DNA typing
Butler, John Marshall, AS-Chemistry (CHEM), University of Virginia
McCord, Bruce, Forensic Science Research and Training Center, Quantico, VA
Bryant, Robert, AS-Chemistry (CHEM), University of Virginia
Hunt, Donald, AS-Chemistry (CHEM), University of Virginia
Allen, Ralph O., AS-Chemistry (CHEM), University of Virginia
Capillary electrophoresis has many attributes which are desirable in DNA separations, namely, speed, high resolution, direct quantitation, and complete automation. The goal of this work has been to develop a CE system to rapidly evaluate forensic DNA samples, principally those derived from the polymerase chain reaction. Factors affecting resolution, sensitivity, and precision have been studied in order to obtain a CE system which performs in a rapid, reliable, and reproducible manner. Separations were found to be primarily dependent upon the concentration and length of the cellulose sieving medium. Sequence dependent binding of DNA intercalators is also described.
This work describes several improvements in the separation of DNA fragments by capillary electrophoresis, including (a) new methods for injection of DNA from highly ionic matrices, (b) significant reduction in separation time without diminishing resolution, and (c) stable operating conditions for routine analysis. The use of a single internal standard for precise quantitation of PCR products and dual internal standards for precise sizing and accurate genotyping of PCR-amplified DNA is also shown. Peak area precision of 3% R.S.D. and peak migration time precision of ≤ 0.2% R.S.D. may be obtained with appropriate internal standards and conditions. In addition, direct comparisons to conventional methods of PCR product analysis have been made to demonstrate the validity of the CE results.
PHD (Doctor of Philosophy)
DNA, polymerase chain reaction, capillary electrophoresis
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