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NCJ Number: NCJ 196175   Add to Shopping cart   Find in a Library
Title: DNA Typing Using High Performance Liquid Chromatography
Author(s): James E. Girard ; Joseph Devaney ; Michael Marino
Corporate Author: American University
United States of America
Date Published: 2001
Page Count: 84
Sponsoring Agency: National Institute of Justice
US Department of Justice
Office of Justice Programs
United States of America
Grant Number: 1999-IJ-CX-0033
Sale Source: American University
4400 Massachusetts Ave., NW
Washington, DC 20016
United States of America

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Document: PDF 
Type: Report (Study/Research)
Language: English
Country: United States of America
Annotation: This is a report on the use of high performance liquid chromatography (HPLC) as a rapid DNA sizing/typing method.
Abstract: Currently, gel electrophoresis is the DNA analysis method most commonly used; however, gel electrophoresis is a slow technique that typically takes more than 2 hours to complete; and once the electrophoresis is complete, the results can take days to process. HPLC, on the other hand, has become a well-established technique for the analysis of biopolymers. Various chromatographic methods are used for the separation of single-stranded and double-stranded DNA, including mixed-mode, size-exclusion, affinity, ion-exchange, reverse-phase, and ion-pairing reverse-phase. Liquid chromatography is a method that is useful for the separation of dsDNA; however, it has not been optimized for the separation of STR systems. In this study, the parameters that affect the liquid chromatographic separation of dsDNA with a bp size between 100 and 400 were examined and optimized. Once optimization of the separation parameters was completed, HPLC was used for the sizing/typing of the HUMTHO1 locus. Finally, the HUMTHO1 locus, a mtDNA repeat, and the amelogenine locus were multiplexed by using HPLC. In addition, the sizing method developed can be used for other PCR products. The separation of dsDNA on a HPLC with a DNAsep column was optimized with the use of RE fragments and PCR products with respect to column temperature, flow rate, and percent ACN per minute. The optimal conditions for the highest resolution and fastest separation were a column temperature of 54 degrees C, flow rate of 0.8 ml/min, and 1 percent ACN per minute. With these optimal conditions, the HUMTHO1 locus was separated and sized by using RE fragments as markers. In addition, the amelogenin locus, a dinucleotide repeat in the human mt genome, and the HUMTHO1 locus were simultaneously sized and typed by using RE fragments. 36 tables and 103 references
Main Term(s): Technology transfer
Index Term(s): Chromatography ; Forensics/Forensic Sciences ; Investigative techniques ; DNA fingerprinting ; NIJ final report
   
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https://www.ncjrs.gov/App/Publications/abstract.aspx?ID=196175

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