Department Chair

M. Scott Goodman, Ph.D., Associate Professor of Chemistry

Date of Award

5-2012

Access Control

Campus-Only Access

Degree Name

Forensic Science, M.S.

Department

Chemistry Department

Advisor

Amy McMillan, Ph.D., Associate Professor of Biology

Department Home page

http://chemistry.buffalostate.edu/forensic-science-ms-1

First Reader

M. Scott Goodman, Ph.D., Associate Professor of Chemistry

Second Reader

Joonyeong Kim, Ph.D., Associate Professor of Chemistry

Abstract

Allelic ladders contain all the alleles at a given locus and since the components of the allelic ladder and the sample fragments have the same length and sequence, sizing is very accurate when conducted with an allelic ladder. Allelic ladders are therefore very useful in population genetics studies. For this study, an allelic ladder for the Bald Eagle, Haliaeetus leucocephalus, microsatellite locus Hal01 was constructed. Consecutive alleles at the Hal01 locus were PCR amplified and analyzed using capillary electrophoresis with fluorescent detection of fragment sizes. The criteria for the selected allele samples were high peak intensity and specificity, and low stutter peak percentage. The selected amplified alleles were then co-amplified to obtain all the alleles in approximately equal amounts. Due to a high stutter percentage observed in Bald Eagle alleles at this locus, optimizing the PCR conditions and reagent concentrations was an important step of the study. The co-amplification reactions were carried out with two different DNA polymerases and using final MgCl2 concentrations of 1.5 mM and 2.0 mM. Co-amplification carried out using Platinum Taq DNA polymerase and 2.0 mM final MgCl2 concentration provided the most ideal allelic ladder. The allelic ladder includes alleles with lengths 137, 141, 143, 145, 149, and 151 bp. The constructed ladder was used to size known alleles. The known alleles lined up correctly with the allelic ladder and hence the ladder is usable for population genetics studies for this locus. During the initial amplification of alleles, an error in allele identification was revealed where all the samples previously identified as allele 139 actually contained allele 141. Due to the ease and reproducibility of the approach of this study, allelic ladders can be constructed for other wildlife species.

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