72
FENG QIAN AND GREGORY G. GERMINO
size. There are, however, a number of other factors that also affect rates of
DNA migration.
DNA conformation The relationship between size and migrationrate is more complicated when
the DNA molecules arenot linear or are single-stranded. Closed circular
(form I), nicked circular (form II), and linear duplex (form III) DNA of
the same molecular weight migrate at different rates. The rate of migration
for a circular molecule depends on the degree of its superhelicity. Closed
circular DNA, which is typically negatively supercoiled, migrates faster than
linear or nicked circular DNA. Conversely, a nicked circular molecule migrates more slowly than a linear one for any given length. Although the
mobility of all the fragments is reduced when ethidium bromide, the intercalating dye, is incorporated into the gel, this effect is most pronounced with
negatively supercoiled DNA. The dye reduces negative superhelicity in the
form I molecule and the radii of the molecules increase, which results in
decreased mobility. With increased concentration of ethidium bromide,
all negative superhelical forms relax. As still more ethidium bromide is
added, positive superhelical turns are generated, the DNA molecules become more compact and their mobility increases rapidly.
Single-stranded molecules (DNA and RNA) typically have considerable secondary structure in solution. The degree of secondary structure depends on
a number of factors including temperature, base composition and buffer
composition/concentration. The variability of secondary structure and
its effects on electrophoretic mobility precludes accurate comparison of
fragment lengths. Therefore, fragment lengths of single-stranded molecules
(DNA or RNA) are determined by electrophoresis on denaturing gels (e.g.,
DNA sequencing gel or Northern blot gel) which eliminate secondary structure.
Applied voltage At low voltage, the rates of migration of short ( <2000 bp) and long linear
DNA fragmentsareproportional to their lengths. This linear relationship is
not valid for very long fragments subjected to a high voltage, however, and
the resolution oflarger fragments decreases when separated by high voltage.
This effect is most pronounced in pulsed field gel electrophoresis (see below).
Matrix The sieving properties of a gel depend on both the composition and concomposition centration of its matrix.
FENG QIAN AND GREGORY G. GERMINO
size. There are, however, a number of other factors that also affect rates of
DNA migration.
DNA conformation The relationship between size and migrationrate is more complicated when
the DNA molecules arenot linear or are single-stranded. Closed circular
(form I), nicked circular (form II), and linear duplex (form III) DNA of
the same molecular weight migrate at different rates. The rate of migration
for a circular molecule depends on the degree of its superhelicity. Closed
circular DNA, which is typically negatively supercoiled, migrates faster than
linear or nicked circular DNA. Conversely, a nicked circular molecule migrates more slowly than a linear one for any given length. Although the
mobility of all the fragments is reduced when ethidium bromide, the intercalating dye, is incorporated into the gel, this effect is most pronounced with
negatively supercoiled DNA. The dye reduces negative superhelicity in the
form I molecule and the radii of the molecules increase, which results in
decreased mobility. With increased concentration of ethidium bromide,
all negative superhelical forms relax. As still more ethidium bromide is
added, positive superhelical turns are generated, the DNA molecules become more compact and their mobility increases rapidly.
Single-stranded molecules (DNA and RNA) typically have considerable secondary structure in solution. The degree of secondary structure depends on
a number of factors including temperature, base composition and buffer
composition/concentration. The variability of secondary structure and
its effects on electrophoretic mobility precludes accurate comparison of
fragment lengths. Therefore, fragment lengths of single-stranded molecules
(DNA or RNA) are determined by electrophoresis on denaturing gels (e.g.,
DNA sequencing gel or Northern blot gel) which eliminate secondary structure.
Applied voltage At low voltage, the rates of migration of short ( <2000 bp) and long linear
DNA fragmentsareproportional to their lengths. This linear relationship is
not valid for very long fragments subjected to a high voltage, however, and
the resolution oflarger fragments decreases when separated by high voltage.
This effect is most pronounced in pulsed field gel electrophoresis (see below).
Matrix The sieving properties of a gel depend on both the composition and concomposition centration of its matrix.