I. Methods for Restriction Endonuclease Studies of DNA Structure. II. Restriction Endonucleolytic Characterization of Animal Mitochondrial DNAs and Human Globin Genes

Author: Parker, Richard Carl

Year: 1979

Degree: Dissertation (Ph.D.)

Advisors: Vinograd, Jerome Rubin; Baldeschwieler, John D.; Maniatis, Tom

Committee Member: Unknown, Unknown

Option: Biochemistry

DOI: 10.7907/pphq-jv67

Abstract

An initial approach to the structural organization of DNA is restriction endonuclease site mapping and gel electrophoretic analysis. This is true for genomes of the simplest or greatest complexities.

Two techniques are presented in this thesis that facilitate this approach. The first uses ethidium bromide to limit the action of a restriction endonuclease on a closed circular DNA in order to derive a set of circularly permuted linear molecules. These molecules, after appropriate treatment, can be used to orient the restriction endonuclease sites and to calibrate the relationship between electrophoretic mobility and DNA fragment size without the introduction of external standards.

The second technique utilizes a low melting temperature agarose. It provides a simple system for two-dimensional electrophoretic analysis of DNA molecules with restriction endonuclease digestion of the DNA occurring after the first electrophoretic separation and before the second.

These techniques and others were used to study mitochondrial DNA from mice and rats. Some of these data explore the evolutionary divergence of the mtDNA in these animals. This information can be compared to evolutionary studies with nuclear DNA.

Additionally, chromosomal DNA from patients with normal hemoglobin and hemoglobin Lepore was studied. Using this type of analysis we were able to demonstrate that a change in the amino acid sequence of some of the β-related globin chains in hemoglobin Lepore is associated with a change in DNA structure.

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