Keywords

Electric fields, Electrophoresis, Fluorescence, Genetics, Molecules

Abstract

This review addresses methods for obtaining sequence information directly from unamplified genomic length DNA. Our generic starting point is a large piece of DNA that contains many thousands of base pairs (kilobase pairs, kbp) or even millions of base pairs (megabase pairs, Mbp). We would like to determine the genomic distance between two repeats of a given sequence, indicated by the red dots on the coiled DNA molecule in Figure 1. Let us consider the case where these sequences are restriction sites, which can be selectively cut using proteins known as restriction enzymes. For decades, gel electrophoresis served as the standard approach for determining the sizes of restriction fragments, with pulsed field gel electrophoresis being the workhorse method when the fragments are longer than tens of kilobase pairs. While the electrophoretic mobility of DNA in free-solution depends on molecular weight only for very short DNA, typically less than around 100 base pairs (bp), there is a strong dependence of electrophoretic mobility on molecular weight when the DNA is forced to move through a porous medium due to the interactions between the DNA and the fibers of the gel. Agarose gels are the medium of choice for double-stranded DNA, with pore sizes in the hundreds of nanometer range. The sizes of the fragments are obtained by comparing their electrophoretic mobilities to known standards (often called a “ladder” with reference to the appearance of regularly spaced bands in a gel) and a theory for interpolating electrophoretic mobilities of DNA fragments in the sample that are between the bands in the ladder.

Original Publication Citation

Chem. Rev. 2013, 113, 4, 2584–2667

Document Type

Peer-Reviewed Article

Publication Date

2012-11-12

Publisher

American Chemical Society

Language

English

College

Ira A. Fulton College of Engineering

Department

Chemical Engineering

University Standing at Time of Publication

Assistant Professor

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