Recombinant DNA Technology

Further Historical Perspective

Geneticists have known for a long time how to isolate DNA from cells.

Geneticists have known for a long time how to chop DNA into small pieces.

What geneticists did not know how to do until the early 1970s was to replicate small fragments of DNA.

1970s Breakthrough

• The discovery of the restriction enzyme

(or restriction endonuclease).

Properties of RE

• Cut double-stranded DNA at specific target sites.

• Allow fragments of DNA that have been cut with the same RE to be rejoined.

AnimationAnimation

• Recombinant DNA_Animation_1

• Recombinant DNA_Animation_2

The joining of two DNA fragments by DNA ligase produces a recombinant DNA molecule

Therefore, eukaryotic DNA could be propagated in prokaryotic cells.

A great breakthrough!!!!!

•

Carriers of foreign DNA are Vectors:

• Most carriers are

• 1. Plasmids

• 2. Bacteriophages (viruses)

• A bacteriophage is a virus that

infects a bacteria.

Introduction to PCR• PCR (polymerase chain reaction)

• PCR is a means to enhance/replicate the amount of DNA collected (in vitro)

* p r c

PCR Creates more DNA for Study

• PCR Animation

DNA Replication Review:

• Add DNA polymerase, all 4 DNA building blocks ???

5’ CTGACGCTGCTGCATGCTAGCT 3’

3’ GACTACGACGACGTACGATCGA 5’

DNA Replication Review:

• Primers are required:

5’ CTGACGCTGCTGCATGCTAGCT 3’

CGA 5’

5’ CTG

3’ GACTACGACGACGTACGATCGA 5’

DNA Replication Review:

• Primers are required:

5’ CTGACGCTGCTGCATGCTAGCT 3’

. . . t a c g a t CGA 5’

5’ CTG a t g c t g . . . .

3’ GACTACGACGACGTACGATCGA 5’

Introduction to Agarose Gel Electrophoresis

Weigh out ~ a gram of agarose.

• Mix the agarose with 50- 100 ml of buffer.

• Heat to dissolve the agarose.

• Assemble the gel tray and comb.

Pour the gel.

• Pick up the DNA sample with a micro-pipettor.

• Load one DNA sample into each well on the gel.

Connect the gel to a low voltage power supply.

After completion of the run, add a DNA staining material and visualize the DNA

under UV light.

• Analyze the results.

• Gel Electrophoresis – demonstration

• Ensure you’re plugged into the web ;)

• Electrophoresis Demo 1 – flash simulation

• Electrophoresis Demo 2 – java applet

• You need to be hooked to the web for these to work ;)