Chapter 5Chapter 5

Resolution and Detection of Nucleic Acids

ObjectivesObjectives

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 ElectrophoresisGel Electrophoresis

Electrophoresis is the movement of molecules by an electric current.

Nucleic acid moves from a negative to a positive pole.

Gel ElectrophoresisGel 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.

500 bp

200 bp

50 bp

% agarose: 2% 4% 5%

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.

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

Types of PFGETypes 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 Polyacrylamide Gel Electrophoresis (Electrophoresis (PAGEPAGE))

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)Capillary Electrophoresis (CE)

Separates solutes by charge/mass ratio.

Capillary gel electrophoresis is used to separate nucleic acids.

=+ + +

+

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Capillary Gel Electrophoresis Capillary Gel Electrophoresis (CGE)(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 BuffersElectrophoresis 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 EquipmentElectrophoresis Equipment

Horizontal or submarine gel

Electrophoresis EquipmentElectrophoresis Equipment

Vertical gel

Electrophoresis EquipmentElectrophoresis 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 GelRunning 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 GelRunning a Gel

Load sample mixed with tracking dye (dye + density agent).

Running a GelRunning 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

SummarySummary

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.