Agarose Gel Electrophoresis

Introduction

  • Agarose gel electrophoresis: a procedure used to separate DNA fragments based on their sizes.
  • DNA has many negative electrical charges.
  • A solution containing a mixture of DNA fragments of variable sizes is placed into a small well formed in an agarose gel that has a texture similar to gelatin.
  • An electric current causes the negatively-charged DNA molecules to move towards the positive electrode.
  • The gel has tiny pores that allows small particles to move through it very quickly and larger particles to move slowly.
  • After a period of exposure to the electrical current, the DNA fragments will sort themselves out by size.
  • Fragments that are the same size will tend to move together through the gel and form bands.

Electrophoretic Analysis of Restriction Fragments

  • Restriction enzymes can be used to create certain DNA fragments by making cuts at the specific sequence of base pair that it recognizes.
  • The three-dimensional shape of a restriction enzyme allows it to fit perfectly in the groove formed by the two strands of a DNA molecule.
  • When attached to the DNA, the enzyme slides along the double helix until it recognizes a specific sequence of base pairs, which signals the enzyme to stop sliding.
  • The enzyme then chemically separates, or cuts, the DNA molecule at that site – called a restriction site.
  • If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments of DNA.
  • Electrophoresis: to carry with electricity
  • Agarose gel electrophoresis separates DNA fragments by size.
  • DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution.
  • A direct current is passed between wire electrodes at each end of the chamber.
  • Since DNA fragments are negatively charged, they will be drawn toward the positive pole (anode) when placed in an electric field.
  • The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger one.
  • The rate at which a DNA fragment migrates through the gel in inversely proportional to its size in base pairs.
  • Smaller DNA fragments will travel farther than larger ones.
  • Fragments of the same size stay together and migrate in single bands of DNA.
    • These bands will be seen in the gel when the DNA is stained.

Making DNA Visible

  • DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis.
  • A loading dye containing several blue dyes is added to the DNA solution.
  • The loading dye does not stain the DNA itself, but makes it easier to load the gels and monitor the progress of the DNA electrophoresis.
  • The dye fronts migrate toward the positive end of the gel, just like the DNA fragments.
  • This dye indicates how far the DNA within the gel has progressed.