Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor's Name

Bruce McCord

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Yong Cai

Third Advisor's Name

Watson Lees

Fourth Advisor's Name

Yuan Liu

Fifth Advisor's Name

DeEtta Mills

Keywords

Pressure Cycling Technology, Differential Extraction, Rape Kits, Sperm DNA Extraction, Alkaline Lysis

Date of Defense

7-3-2014

Abstract

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures.

Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile.

Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

Identifier

FI14071167

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