Part 1. Gel electrophoresis

• DNA is negatively charged (because of phosphate backbone)

• DNA will be attracted to positively charged poles and repelled from negatively charged ones

Part 2. Restriction Endonucleases (aka restriction enzymes)

• Enzymes that “cut” DNA in a sequence-specific manner

• Serve as a natural defense mechanism for bacteria against viral infection

• Bacteria protect their DNA from cutting by their own enzymes through methylation

Examples

Enzyme Recognition sequence • EcoRI GAATTC• HindIII AAGCTT• BamHI GGATCC• EcoRV GATATC

• Recognition sequences are usually 4-8 base pairs in length and are usually palindromic

A closer look…. BamHI

5’….ACTGTACGGATCCGCTA….3’3’….TGACATGCCTAGGCGAT….5’

BamHI

A closer look…. BamHI

5’….ACTGTACG GATCCGCTA….3’3’….TGACATGCCTAG GCGAT….5’

Ligations

• When DNA molecules with sticky ends come together, only hydrogen bonds between complimentary nucleotides are reformed

• These H-bonds are not stable enough to be permanent

• DNA ligase=enzyme that joins the ends of DNA and re-establishes the phosphodiester bond in the DNA molecule

5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

DNA Ligase

Running a gel

• Molten agarose is poured into a casting tray and a comb is placed

• After the agarose solidifies, the comb is removed leaving wells where the DNA will be loaded

• DNA samples are mixed with tracking dye which contains sucrose (to weigh down the DNA) and dyes so that you can visualize migration

• A buffer containing ions (to conduct an electric current) is placed in the chamber around the gel

Gel electrophoresis

Agarose gel

- electrode + electrodeDNA fragments

buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

Gel electrophoresis

- electrode + electrode

current

buffer~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

~~~~~~~~~~~~~~~~~~~~~~~~

Movement of DNA fragments in agarose gels

• There is a linear relationship between the migration rate of a given DNA fragment and the logarithm of its size (in basepairs).

• Larger molecules move more slowly through the gel because of more friction

An ethidium-stained gel photographed under UV light

**Each band that you see is a collection of millions of DNA molecules, all of the same length!!