1. Resolution and Detection of Nucleic Acids
Chapter 5
Resolution and Detection of Nucleic Acids

2. 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.

3. Gel Electrophoresis
Electrophoresis is the movement of molecules by an electric current.
Nucleic acid moves from a negative to a positive pole.

4. 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.

5. 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% % %
500 bp
200 bp
50 bp
500 bp
200 bp
50 bp
500 bp
200 bp
50 bp
The concentration of gel/buffer will affect the resolution of fragments of different size ranges.

6. Gel Electrophoresis
Slab gel electrophoresis can have either a horizontal or vertical format.
Sample is introduced into wells at the top of the gel.

7. Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE).

8. 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

9. 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)

10. Capillary Electrophoresis (CE)
Separates solutes by charge/mass ratio.
Capillary gel electrophoresis is used to separate nucleic acids. =
+ + + ++ -

11. Capillary Gel Electrophoresis (CGE)
Thin glass (fused silica) capillary 30 to 100 cm X 25–100 mm 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

12. 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)

13. Electrophoresis Equipment
Horizontal or submarine gel

14. Electrophoresis Equipment
Vertical gel

15. 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

16. 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.

17. Running a Gel
Load sample mixed with tracking dye (dye + density agent).

18. 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

19. 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.