1. Part 3

2. Mutagen
Gene
transfer
Genetic
recombi
nation
Selection of mutants

3. Recombinant DNA technology, gene cloning

4. • Genetic engineering involves changing
the genetic material in an organism to
alter its traits or products
• A recombinant DNA molecule contains
DNA fragments spliced together from 2 or
more organisms

5.  Stages of cloning experiment
 Elements:
 Vectors
 Restriction enzymes
 Mechanism in joining the fragments
 Selection or detection of successful cloning
 Gene library

6. 1. Joining
DNA segment
Vector 2. Providing
milieu that
allows
propagation
Clones

7. 1. Remove bacterial DNA (plasmid).
2. Cut the Bacterial DNA with
“restriction enzymes”.
3. Cut the DNA from another
organism with “restriction
enzymes”.
4. Combine the cut pieces of DNA
together with another enzyme and
insert them into bacteria.
5. Reproduce the recombinant
bacteria.
6. The foreign genes will be expressed
in the bacteria.

8. 1. It must be able to replicate
2. There must be some way to introduce
vector DNA into the cell
3. There must be some way of detecting its
presence, preferably by plating techniques
Most common vectors are:
 Plasmid, phage λ and viruses

9.  Discovered in an experiment where
bacteriophage lost its plaque formation in
E.coli
 Enzymes that recognize a specific base
sequence in a DNA molecule
 It makes two cuts, one in each strand,
generating 3’-OH and 5’-P termini
 Types of termini produced:
 Flush or blunt end
 Cohesive or sticky end

12.  The number of cuts made in the DNA from
specific organism is limited
 A particular restriction enzyme generates a
unique family fragments from a DNA
molecule

13. •BstEII pUC19 pUC19 •HindIII •HindIII •BstEII

14. Restriction maps show the relative location of a selection of restriction sites along
linear or circular DNA.
EcoRI
HindIII
PstII BamHI
HindIII BamHI PstII

15. Digests
1: EcoRI
2: HindIII
3: EcoRI + HindIII
Resultant Fragments: approximate sizes
1: 3 kb, 5 kb
2: 6 kb, 2 kb
3: 2 kb, 1 kb, 5 kb,

16. BglII BglII BamHI
BglII BamHI PstI +BamHI +PstI +PstI
5.2
4.2
3.6 3.5
3.3
2.6
1.7 1.7
1.4 1.4
1.2 1.2 1.2
1.0 0.9 1.0
0.7
0.5
0.3 0.3 0.3
BglII BamHI PstI BglII PstI
0.3 0.7 2.6 0.9 0.5 1.2

17. A) 11, 6, 5
B) 14,8
C) 16,6
A x B) 8, 6, 5, 3
A x C) 11, 5, 5, 1
B x C) 8, 8, 6

18.  Cohesive end
 Blunt end
 Increasing the DNA concentration and addition of
ligase
 Addition of homopolymers
 Using of linkers

22.  Antibiotic resistance marker
 Insertional inactivation
 Electrophoresis
 hybridization

25.  collection of all of the vector molecules, each
carrying a piece of the chromosomal DNA of
the organism