Download All Living things pass on their genetic heritage by common

All Living things pass on their genetic heritage by common processes.
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
EcoRI:
BamHI:
HindIII
Target sequences
“Sticky end” products

GAATTC
CTTAAG
G
CTTAA
AATTC
G
CCTAG
GATCC
A
TTCGA
AGCTT

GGATCC
CCTAGG

AAGCTT
TTCGAA



G
G
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.
xxxxxxxxxxxG
xxxxxxxxxxxCTTAA
Gxxxxxxxxxxxxxx
AATTC.........human.............G
AATTCxxxxxxxxxxxxxx
G........human..............CTTAA
 DNA ligase
xxxxxxxxxxxxxxxxxxxxxxxxxGAATTC........human.................GAATTCxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxCTTAAG.........human................CTTAAGxxxxxxxxxxxxxxxxxxxxxxxxxxx
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
1
2
1
G*
2
1
2
G* terminated:
1
2
3 4
G*
3 4
3 4
1
2
3 4
1
2
C*
3
1
2
C* terminated:
6
7
8
6
7
8
3 4
5
6
7
C*
8
6
T*
7
1
2
3 4
T*
5
1
2
3 4
5
A*
10
9
8
7
6
5
4
3
2
1
5
5
T* terminated:
Sequencing Gel
5
A*
6
G*
C*
G*
9
9
G*
10
T*
Cathode (-)
Mobility

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
___
B
___
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
C
D
___
___
A
B
C
___
___
___
___
___
___
D
___
___
___
___
Relationship between species
A, B, C = Closely related
D = Less closely related
A
___
B
___
C
___
D
___
___
___
___
___
___
___
___
___
___
___
___
___
___