Molecular Genetics Lab

Review

Bacterial Transformation• Genetic transformation—host organism takes

in and expresses foreign DNA• Genetic engineering—manipulation of an

organism’s genome using DNA technology– Introduction of foreign DNA into organism

• You used antibiotic-resistance plasmids to transform E. coli– If bacteria incorporates the foreign

DNA, they became ampicillin resistant

E. coli

• Most common bacterium in human gut• Studied extensively• Important research organism– Reproduce rapidly– A single cell can divide to form millions of cells

overnight– No nuclear envelope surrounding chromosome– One single chromosome– Some contain plasmids resistant to certain drugs

Plasmids

• Circular pieces of DNA• Outside the main bacterial chromosome• Carry their own genes for specialized functions• Plasmids can be used in genetic engineering to

introduce foreign genes into a bacterium

The Experiment

Results

Gel Electrophoresis

Restriction Enzymes

• Bacteria have enzymes that cut (or digest) the DNA of foreign organisms to protect themselves

• Scientists have isolated several hundred of these enzymes, restriction enzymes

• Each is able to recognize and cut at a specific DNA sequence, the recognition sequence

• DNA fragments can be analyzed and used

The Experiment

• You used restriction enzymes to cut DNA into fragments

• You used gel electrophoresis to separate the samples of DNA that had been cut

• You compared fragments of unknown size to fragments of a known size to calculate the unknown fragment sizes

Restriction Enzymes

• Restriction enzymes are specific to an exact recognition sequence

• Sticky ends & Blunt ends

Gel Electrophoresis

• Separates molecules of the rate of movement through a gel under electricity

• DNA is negatively charged—will move toward positive pole of the gel

• Different sized fragments move at different rates– Smallest move the most quickly, thus migrate the

farthest

Analysis of Results

• Each fragment is a particular number of nucleotides long

• To find out the size of the unknown DNA fragments, you ran it along side DNA with known fragment sizes

• HindIII cut DNA into known fragment sizes• It will determine fragment sizes of DNA cut by

EcoRI

Graphing HindIII