Semester

Spring

Date of Graduation

2012

Document Type

Thesis

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Jeffrey Wells

Committee Co-Chair

Clifton Bishop

Committee Member

Keith Morris

Abstract

STR loci consist of short, repetitive sequence elements 2--7 base pairs in length. An STR genotype is typically determined by PCR amplification followed by fragment size estimation with electrophoresis. Commercial multiplex STR amplification kits are available for the 13 CORE loci used in the USA for human identity testing. The genotyping rationale with these multiplex STR amplification kits involves a two-fold calibration analysis: 1) An internal size standard (ISS) labeled with a separate dye establishes the size of the unknown DNA fragment and 2) an allelic ladder which contains most of the common alleles for each locus is also sized by the internal sized standard and used as a reference for the allele call. Locus-Specific Brackets (LSBs) offer an alternative STR sizing method that may be less susceptible to the problems caused by variation in electrophoretic conditions. An LSB multiplex may also be cheaper to produce and use than a conventional kit. LSBs are internal size standards that flank each targeted locus. The brackets have the same repeat structure as the locus, are 1 or more repeat units shorter or longer than the common alleles, and are labeled with the same dye. Because LSBs have the same electrophoretic properties as the sample alleles, their sizing function should not be impaired by injection-to-injection variation in conditions. The aim of this project was to produce an LSB-based human STR multiplex reaction kit, free of allelic dropout and with appropriate LSB size standards for analysis by custom genotyping software, and to do a simple performance evaluation of the kit by testing the reproducibility of profiles and the sensitivity of the reaction. The result was a new human STR multiplex that can amplify all 13 STR loci plus amelogenin in a single reaction. The new LSB multiplex has a sensitivity range of 0.1-1ng of DNA template where full genetic profiles can be produced. Also developed were new locus specific brackets needed to accommodate necessary shifts allele size range. Existing software failed to successfully genotype all alleles; however manual analysis of the data showed the correct genotypes for 81% of all tested alleles, with few incorrect calls as fragment were incorrectly sized by the size standard. Although the further development of the kit is clearly required, the LSB kit remains promising as a new and alternative method for forensic DNA analysis.

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