Separation and Detection of Nucleic Acids via Electrophoresis Methods

Analysis of a DNA sequence can be accomplished via a method called electrophoresis. Electrophoresis is a term that basically describes the movement of molecules by way of an electric current and separation of those molecules based on size. This process occurs through agarose or polyacrylamide gels, which serve as a way to limit migration of molecules as they move from the negative anode to positive anode. Small molecules move through the gel matrix faster than larger molecules.

How does it work?

Each phosphate group from a DNA molecule is ionized

DNA becomes negatively charged

DNA migrates towards the positive pole (anode)

 

Factors Affecting Electrophoretic Separation

  • Ÿ Strength of the electric current (voltage)
  • Ÿ Concentration and type of the buffer
  • Ÿ Gel density
  • Ÿ Size of the DNA
AGAROSE CONCENTRATION AND SEPARATION RANGES
Agarose Concentration (%) Separation Range (base pair size)
0.3 5,000 – 60,000
0.6 1,000 – 20,000
0.8 800 – 10,000
1.0 400 – 8,000
1.2 300 – 7,000
1.5 200 – 4,000
2.0 100 – 3,000

As agarose concentration increases, the separation range decreases

 

TYPES OF ELECTROPHORESIS SYSTEMS
Pulsed Field Electrophoresis
  • Ÿ Best for very large DNA molecules
  • Ÿ Current is applied in alternating directions
Field Inversion Gel Electrophoresis

(FIGE)

  • Ÿ Alternates the + and – electrodes
  • Ÿ Requires temperature controls and a switching mechanism
Polyacrylamide Gel Electrophoresis

(PAGE)

  • Ÿ Best for very small DNA fragments
  • Ÿ Initially used for protein separation, but can also be used for high resolution of nucleic acids
Capillary Electrophoresis
  • Ÿ Molecules are separated by size and charge
  • Ÿ Small Molecules = Fast
  • Large Molecules = Slow
  • Negatively Charged = Fast
  • Positively Charged = Slow
  • Ÿ Utilizes a polymer inside of a capillary instead of a gel
  • Ÿ Increased sensitivity

Understanding Buffer Systems

In order to change the pH of a buffered solution by one point, either the acidic or basic form of the buffer must be brought to a concentration 1/10th that of the other form.

Test Your Knowledge

  1. Based on the diagram, determine the sizes (to the best approximation) of the DNA fragments for each of the samples:

 

 

electro1

Answer:

  • Sample A: 200bp, 700bp
  • Sample B: 90bp, 600bp, 875bp
  • Sample C: 400bp

 

L Noll Image_small

-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

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