Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Resolution and Detection of Nucleic Acids
Chapter 5
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Gel Electrophoresis
 Electrophoresis is the movement of molecules by an electric current.
 Nucleic acid moves from a negative to a positive pole.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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
50 bp
200 bp
50 bp
50 bp
The concentration of gel/buffer will affect the resolution of fragments of different size ranges.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Gel Electrophoresis
 Slab gel electrophoresis can have either a horizontal or vertical format.
 Sample is introduced into wells at the top of the gel.
 Because each nucleotide has one negative charge, the charge‐
to‐mass ratio of molecules of different sizes will remain constant. DNA fragments will therefore migrate at speeds inversely related to their size.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Very large DNA molecules are separated by pulsed field gel electrophoresis (PFGE).
Very large pieces (50,000–250,000+
bp) of DNA cannot be resolved
efficiently by simple agarose
electrophoresis. Even in the lowest
concentrations of agarose,
megabase fragments are too
severely impeded for correct
resolution.
Pulses of current applied to the gel
in alternating dimensions to
enhance migration.
http://www.bio.davidson.edu/courses/genomics/method/pulse_field.html
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition

Types of PFGE
Field inversion gel electrophoresis (FIGE): alternating positive and negative poles

In this type of separation, the DNA goes periodically forward and backward. FIGE requires temperature control and a switching mechanism.

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
Copyright © 2012 F.A.
Davis Company
http://www.nal.usda.gov/pgdic/Probe/v2n3/puls.html
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Application  Bacterial typing for epidemiological purposes ‐ require the resolution of chromosome‐sized fragments of DNA.  Enzymatic digestion of genomic DNA will yield a set of fragments that produce a band pattern specific to each type of organism. By comparing band patterns, the similarity of organisms isolated from various sources can be assessed. This information is especially useful in determining the epidemiology of infectious diseases, for example, identifying whether two biochemically identical isolates have a common source.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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
 APS produces free oxygen radicals in the presence of TEMED to drive the polymerization mechanism. Excess oxygen inhibits the polymerization process. Therefore, deaeriation, or the removal of air, of the gel solution is done before the addition of the nucleating agents (APS & TEMED).
 Resolves 1 bp difference in a 1 kb molecule (0.1% difference)
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
composition of polyacrylamide gels  The composition of polyacrylamide gels is represented as the total percentage concentration (w/v) of monomer (acrylamide with cross‐linker), T, and the percentage of monomer that is cross‐linker, C.  For example, a 6% 19:1 acrylamide:bis gel has a T value of 6% and a C value of 1/20, or 5%.
 Increasing T decreases the pore size proportionally. The minimum pore size (highest resolution for small molecules) occurs at a C value of 5%. Variation of C above or below 5% will increase pore size. Usually, C is set at 3.3% (29:1 ) for native and 5% (1 9:1 ) for standard DNA and RNA gels.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Advantage  Higher resolution capability of polyacrylamide for small fragments.
 The components of polyacrylamide gels are synthetic; thus, there is not as much difference in batches obtained from different sources.
 Altering T and C in a polyacrylamide gel can change the pore size and, therefore, the sieving properties in a predictable and reproducible manner.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Capillary Electrophoresis (CE)
 Separate organic chemicals (pharmaceuticals and carbohydrates) and inorganic anions and metal ions.
 Separates solutes by charge/mass ratio.
 Negatively charged molecules are completely ionized at high
pH, whereas positively charged solutes are completely protonated in low pH buffers.
 Faster and cheaper than HPLC
 Capillary gel electrophoresis is used to separate nucleic acids.
Copyright © 2012 F.A. Davis Company
+
+
+
+
+
+
-
=
=
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Capillary Gel Electrophoresis (CGE)
 Thin glass capillary 30–100 cm x 25–100 m internal diameter ‐

fused silica ‐ allowing for the passage of fluorescent light
 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
Electro‐kinetic injection of sample
 Capillary electrophoresis separates particles by size (small, fast migration; large, slow migration) and charge (negative, fast migration; positive, slow migration).
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Electrophoresis Buffers
 Buffers carry current and protect samples during electrophoresis.
 Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), and Tris Phosphate EDTA (TPE) are used most often for DNA.
 TBE has a greater buffering capacity and will give sharper resolution than TAE buffer. TBE is generally more expensive than TAE, and inhibits DNA ligase which may cause problems if subsequent DNA purification and ligation steps are intended.
 10 mM sodium phosphate or MOPS buffer is used for RNA.
 Buffer additives modify sample molecules.
 Formamide, urea (denaturing agents)
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Electrophoresis Equipment
Horizontal or submarine gel
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Electrophoresis Equipment
Vertical gel Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Gel Electrophoresis Requirements
 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.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Sample Loading
Load sample mixed with tracking dye (dye + density agent).
Tracking Dye Comigration
Bromophenol
Xylene Cyanol
Gel %
Agarose
0.5–1.5
2.0–3.0
4.0–5.0
Blue (Nucleotides)
(Nucleotides)
300–500
80–120
20–30
4000–5000
700–800
100–200
PAGE
4
6
8
10
12
20
95
60
45
35
20
12
450
240
160
120
70
45
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Detection  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
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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.
Copyright © 2012 F.A. Davis Company