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Chapter 5

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.

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Chapter 5

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  1. 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% 4% 5% 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.

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