Chapter 5

Resolution and Detection of Nucleic Acids

Objectives
 Explain the principle and performance of electrophoresis
as it applies to nucleic acids.
 Compare and contrast agarose and polyacrylamide gel
polymers.
 Explain the principle and performance of capillary
electrophoresis as it is applies to nucleic acid
separation.
 Describe the general types of equipment used for
electrophoresis.
 Discuss methods and applications of pulsed field gel
electrophoresis.
 Compare and contrast detection systems used in
nucleic acid applications.
Gel Electrophoresis
 Electrophoresis is the movement of
molecules by an electric current.

 Nucleic acid moves from a negative to a

positive pole.
Gel Electrophoresis
 When DNA is applied to a macromolecular cage or gel
such as agarose or polyacrylamide, its migration under
the pull of the current is impeded.
The movement of molecules is impeded in the gel so that
molecules will collect or form a band according to their
speed of migration.
% agarose: 2% 4% 5%

500 bp
500 bp
200 bp
200 bp

500 bp

200 bp 50 bp

50 bp
50 bp

The concentration of gel/buffer will affect the resolution of

fragments of different size ranges.
Gel Electrophoresis
 Slab gel electrophoresis can have either a
horizontal or vertical format.

 Sample is introduced into wells at the top

of the gel.
Very Large DNA Molecules are Separated by
Pulsed Field Gel Electrophoresis (PFGE).
Types of PFGE
 Field inversion gel electrophoresis (FIGE):
alternating positive and negative poles
 Transverse alternative field electrophoresis
(TAFE): transverse angle reorientation of poles
on a vertical gel
 Contour-clamped homogenous electric field
(CHEF): alternating polarity in an electrode
array
 Rotating gel electrophoresis (RGE): rotating gel
with fixed poles
Polyacrylamide Gel
Electrophoresis (PAGE)
 Acrylamide, in combination with a cross
linker, methylene bis-acrylamide
 Synthetic, consistent polymer
 Polymerization catalysts: ammonium
persulfate (APS) plus N,N,N',N'-
tetramethylethylenediamine (TEMED), or
light activation
 Resolves 1 bp difference in a 1 kb
molecule (0.1% difference)
Capillary Electrophoresis (CE)
 Separates solutes by charge/mass ratio.

+
+
+ + + -
- =

+
=

 Capillary gel electrophoresis is used to separate nucleic

acids.
Capillary Gel Electrophoresis
(CGE)
 Thin glass (fused silica) capillary 30 to 100
cm X 25–100 m internal diameter
 Linear or cross-linked polyacrylamide or
other linear polymers used for sieving
 Separation based on size
 More rapid, automated than slab gels
 Run at higher charge per unit area
 Electrokinetic injection of sample
Electrophoresis Buffers
 Carry current and protect samples during
electrophoresis.
 Tris Borate EDTA (TBE), Tris Acetate EDTA
(TAE), Tris Phosphate EDTA (TPE) used most
often for DNA.
 10 mM sodium phosphate or MOPS buffer used
for RNA.
 Buffer additives modify sample molecules.
 Formamide, urea (denaturing agents)
Electrophoresis Equipment
Horizontal or submarine gel
Electrophoresis Equipment
Vertical gel
Electrophoresis Equipment
Combs are used to put wells in the cast gel for
sample loading.

 Regular comb: wells separated by an “ear” of gel

 Houndstooth comb: wells immediately adjacent

Running a Gel
 Use the proper gel concentration for
sample size range.
 0.5–5% agarose
 3.5–20% polyacrylamide
 Use the proper comb (well) and gel size.
Running a Gel
Load sample mixed with tracking dye
(dye + density agent).
Running a Gel
 Detect bands by staining during or after
electrophoresis
 Ethidium bromide: for double-stranded
DNA
 SyBr green or SyBr gold: for single- or
double-stranded DNA or for RNA
 Silver stain: more sensitive for single- or
double-stranded DNA or for RNA and
proteins
Summary
 Electrophoresis is used to separate molecules
by size and/or charge.
 Nucleic acid fragments can be resolved on
agarose of polyacrylamide gels.
 PFGE is used to resolve very large DNA
fragments.
 CGE is more rapid and automated than slab gel
electrophoresis.
 The choice of electrophoresis method depends
on the type and size of sample.