Strategies for low copy number DNA analysis

Keywords

DNA -- Analysis; Forensic genetics -- Technique; Gene amplification; Polymerase chain reaction

Abstract

Low copy number (LCN) DNA analysis is an approach that involves the examination of minute quantities (i.e.,

multiple displacement amplification (MDA). PEP PCR uses a mixture of random 15-base oligonucleotide primers to prime and subsequently amplify the whole genome (or a large percentage of it) prior to subsequent genetic analysis while DOP PCR uses a single degenerate primer. MDA is an isothermal process that employs random hexamer primers to amplify the whole genome. Epithelial and sperm cell suspensions were subjected to PEP PCR, DOP PCR, and MDA. Aliquots of the products were re-amplified using primers specific for the D 1 S80 PCR VNTR locus. Cycle numbers ranged from the standard 30 cycles up to 50 cycles. Results indicated that WGA products from PEP PCR and DOP PCR could be used to analyze <100 pg of DNA and to increase the sensitivity of D1S80 analysis from a few cells to one cell equivalent when the cycle number used for DI S80 analysis was increased above the standard cycle number. Although WGA products from MDA could be used to type a few ( <5) cells, the sensitivity of D 1 S80 analysis was not increased using this WGA method. Allelic drop-out and non-specific amplification were observed with each WGA method tested. The preceding LCN DNA typing strategies used the D1S80 PCR VNTR system as a model. The same strategies are being applied to Y-STR typing systems and the efficacy of these methods for forensic casework specimens is being examined.

Notes

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Graduation Date

2003

Advisor

Ballantyne, Jack

Degree

Master of Science (M.S.)

College

College of Arts and Sciences

Department

Chemistry

Format

PDF

Pages

135 p.

Language

English

Length of Campus-only Access

None

Access Status

Masters Thesis (Open Access)

Identifier

DP0028721

Subjects

Arts and Sciences -- Dissertations, Academic; Dissertations, Academic -- Arts and Sciences

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