Chapter 5Resolution and Detection of Nucleic Acids

ObjectivesExplain 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 ElectrophoresisElectrophoresis is the movement of molecules by an electric current.

Nucleic acid moves from a negative to a positive pole.

Gel ElectrophoresisWhen 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 bp500 bp

200 bp

50 bpThe 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 PFGEField inversion gel electrophoresis (FIGE): alternating positive and negative polesTransverse alternative field electrophoresis (TAFE): transverse angle reorientation of poles on a vertical gelContour-clamped homogenous electric field (CHEF): alternating polarity in an electrode arrayRotating 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 activationResolves 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 25100 mm internal diameter Linear or cross-linked polyacrylamide or other linear polymers used for sievingSeparation based on size More rapid, automated than slab gelsRun at higher charge per unit areaElectrokinetic injection of sample

Electrophoresis BuffersCarry 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 EquipmentHorizontal or submarine gel

Electrophoresis EquipmentVertical 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 GelUse the proper gel concentration for sample size range.0.55% agarose3.520% polyacrylamideUse the proper comb (well) and gel size.

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

Running a GelDetect bands by staining during or after electrophoresisEthidium bromide: for double-stranded DNASyBr green or SyBr gold: for single- or double-stranded DNA or for RNASilver stain: more sensitive for single- or double-stranded DNA or for RNA and proteins

SummaryElectrophoresis 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.