Lecture Outline No. 15: Genetic Engineering
Gene Cloning
1. Restriction enzymes: Hundreds of protective bacterial enzymes hydrolyze specific palindromic sequences of invading DNA (i.e. viruses or plasmids).
Enzymes Target sequences “Sticky end” products
EcoRI:

GAATTC
CTTAAG


BamHI: GGATCC
HindIII
CCTAGG


AAGCTT
TTCGAA

G
CTTAA
G
CCTAG
A
TTCGA
AATTC
G
GATCC
G
AGCTT
A
2. Formation of recombinant DNA. Two foreign DNA’s (i.e. human and bacterial) hydrolyzed by the same restriction enzyme have complementary, sticky ends which form hybrid, recombinant DNA molecules. sealed by DNA ligase. xxxxxxxxxxx G
 
AATTC......... xxxxxxxxxxx CTTAA


G xxxxxxxxxxxxxx
 human .............G

G........ human ..............CTTAA


AATTCx xxxxxxxxxxxxx

DNA ligase xxxxxxxxxxxxxxxxxxxxxxxxx GAATTC........ human .................GAATTCx xxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxx CTTAAG......... human ................CTTAAG xxxxxxxxxxxxxxxxxxxxxxxxxxx
3. Expressing cloned DNA in bacteria and yeast.
Bacteria and contain plasmids, small circular DNA's, which replicate independently of the chromosome. By making identical cuts in donor DNA containing genes of interest and specially designed plasmids, recombinant DNA molecules are formed. In bacteria or yeast transformed with plasmids, the foreign genes are
transcribed and translated. Yeasts can splice split, eukaryotic mRNA.
4. Detecting bacteria with recombinant plasmids producing proteins of interest.

Transformed bacterial colonies are grown on agar plates.

Bacterial colonies are transferred to a replica, reference plate..

The bacteria on the agar plate are lysed to release proteins in situ.

The proteins are blotted on a nitrocellulose sheet.

Radiolabeled antibodies specific for the protein are added to the lysed colonies.
The plates are exposed to X-ray film to identify the transformed colony.

The DNA is extracted from the cells on the reference plate for analysis.

DNA Sequencing: Sanger base-specific, replication termination.
DNA sequence A G C G T A T C G G
1 2 3 4 5 6 7 8 9 10
A* terminated: A*
1
A*
1 2 3 4 5 6
G* terminated: G*
1 2
G*
1 2 3 4
G*
1 2 3 4 5 6 7 8 9
G*
1 2 3 4 5 6 7 8 9 10
C* terminated: C*
1 2 3
C*
1 2 3 4 5 6 7 8
T* terminated: T*
1 2 3 4 5
T*
1 2 3 4 5 6 7
Sequencing Gel A* G* C* T*
10
9
8
Cathode (-)
7 Mobility
6

5
4
3
2
1 Anode (+)
Note: The negatively charged, sequence fragments are separated by size by an electric current in a gel; the smaller the fragment the faster its mobility in the gel.
Restriction Fragment Length Polymorphisms (RFLP’s)

The lengths of the DNA fragments produced by treating the DNA of different individuals with the same restriction enzymes are frequently different. These differences are referred to as restriction fragment length polymorphisms, (i.e. “riflips).
Detection of RFLP’s by Blot Hybridization.

Cut DNA into fragments with a restriction enzyme.

Separate the fragments in agarose gels using an electric current.

Denature the fragments into single strands.

Blot transfer the fragments from the gel to a nylon membrane.

Incubate the blot with a radioactive hybridization probe.

The positions of the fragments are detected as radioactive bands on X-ray film.
Uses of RFLP’s
Diagnosis of Genetic diseases
A = Normal gene - Individual 1
B = Normal gene - Individual 2
C = Point mutation
D = Deletion
Forensic cases
A = Victim
B = Evidence
C = Guilty suspect
D = Innocent suspect
Relationship between species
A B
___ ___
C
___
A
___
___
B
___
___
___
___
C
___
___
___
D
D
___
___
D
A, B, C = Closely related
D = Less closely related
A B C
___ ___ ___
___ ___ ___
___
___
___
___
___ ___ ___
___
___
___
___