molecular genetics lab review. bacterial transformation genetic transformation—host organism takes...

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