Chapter 4
Electrophoresis of DNA and RNA Fragments
FENG QIAN AND GREGORY G. GERMINO
lntroduction
Characterization ofDNA and RNA samples usuallyinvolves electrophoretic
analysis. This chapter reviews basic principles and applications of electrophoresis, distinguishing between single field and pulsed field techniques.
Also included is a review of the various matrices commonly used in molecular biology (agarose, acrylamide, Hydro-LinkrM). Methods of purification, detection and identification are discussed.
Electrophoresis plays an integral part of most strategies that involve
characterization or manipulation of nucleic acids. The net negative charge
of the phosphate groups along the DNA or RNA backhone results in migration of nucleic acids to the anode when exposed to a voltage gradient. In this
section, we review many of the factors that affect the rate of electrophoretic
migration of nucleic acids and discuss how some of these can be adjusted to
optimize the separation, identification, purification or quantification of nucleic acids.
Parameters affecting the rate of DNA migration
Molecules of linear double-stranded DNA are oriented in an end-on conformation in an electric field and migrate at ratesthat are inversely proportional to the logarithm of their molecular weight. Consequently, the molecular weight of a DNA fragment of unknown size can be determined by
comparing its mobility to that of the molecular weight standard of known
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +01-410-614-1650;fax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)
Molecular size
of the DNA
Electrophoresis of DNA and RNA Fragments
FENG QIAN AND GREGORY G. GERMINO
lntroduction
Characterization ofDNA and RNA samples usuallyinvolves electrophoretic
analysis. This chapter reviews basic principles and applications of electrophoresis, distinguishing between single field and pulsed field techniques.
Also included is a review of the various matrices commonly used in molecular biology (agarose, acrylamide, Hydro-LinkrM). Methods of purification, detection and identification are discussed.
Electrophoresis plays an integral part of most strategies that involve
characterization or manipulation of nucleic acids. The net negative charge
of the phosphate groups along the DNA or RNA backhone results in migration of nucleic acids to the anode when exposed to a voltage gradient. In this
section, we review many of the factors that affect the rate of electrophoretic
migration of nucleic acids and discuss how some of these can be adjusted to
optimize the separation, identification, purification or quantification of nucleic acids.
Parameters affecting the rate of DNA migration
Molecules of linear double-stranded DNA are oriented in an end-on conformation in an electric field and migrate at ratesthat are inversely proportional to the logarithm of their molecular weight. Consequently, the molecular weight of a DNA fragment of unknown size can be determined by
comparing its mobility to that of the molecular weight standard of known
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +01-410-614-1650;fax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)
Molecular size
of the DNA