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Response of short tandem repeat systems to temperature and sizing methods
Brittany Hartzell1, Kylie Graham, Bruce McCord
1Department of Chemistry and Biochemistry, 136 Clippinger Laboratories, Athens, OH 45701, USA.
Forensic Science International
|June 6, 2003
Summary
Temperature variations impact DNA size determination in capillary electrophoresis (CE). The Global Southern method improves reproducibility during STR analysis, especially when using POP 4 buffer, with higher pH minimizing secondary structure effects for precise size estimates.
Area of Science:
- Forensic Science
- Molecular Biology
- Analytical Chemistry
Background:
- Capillary electrophoresis (CE) is sensitive to temperature fluctuations affecting DNA size determination.
- Secondary structures in amplified DNA and internal standards contribute to migration variations.
- STR analysis relies on accurate sizing curves influenced by method choice and temperature.
Purpose of the Study:
- To investigate the impact of temperature and sizing methods on DNA migration reproducibility in CE.
- To compare the effectiveness of Global Southern and Local Southern sizing methods under varying temperatures.
- To evaluate the influence of buffer pH on DNA size estimates and secondary structure effects.
Main Methods:
- Analysis of two fluorescently labeled allelic ladders (CTTv and Y-PLEX 6) using ABI Prism 310 Genetic Analyzer.
- Electrophoresis conducted at temperatures ranging from 35-70°C with POP 4 buffer (pH 8).
- Comparison of Global Southern and Local Southern sizing methods, and evaluation of an experimental buffer (3% hydroxyethylcellulose, pH 11).
Main Results:
- The Global Southern method demonstrated greater robustness against temperature variations compared to the Local Southern method.
- Regression analysis revealed temperature sensitivity in migration times, highlighting the importance of the chosen sizing method.
- Elevated pH (11) in the experimental buffer minimized secondary structure effects, leading to enhanced precision in DNA size estimates.
Conclusions:
- The Global Southern sizing method is recommended for STR analysis in CE when temperature fluctuations are anticipated.
- Optimizing buffer conditions, such as increasing pH, can significantly reduce secondary structure influences on DNA migration.
- These findings contribute to improving the accuracy and reproducibility of DNA sizing in forensic and genetic analyses.