NCJ Number
196175
Date Published
2001
Length
84 pages
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
Date Published: January 1, 2001