3_2021_09_11!12_mmmmmmmmmmmmm06_58_PM.ppt

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1. Molecular Cloning or Genetic Engineering or Recombinant DNA
Technology:
To clone means to make identical copies. DNA cloning involves
separating a specific gene or DNA segment from a larger chromosome,
attaching it to a small carrier DNA. The resultant hybrid DNA is called
recombinant DNA, which is transferred to a proper host (bacteria, virus
or yeast) and replicated to make multiple copy of the selected gene.
When cloned under an appropriate expression vector, a gene can be
expressed (I.e. transcribed and translated), at desired level to produce
recombinant proteins.
This technology has made it possible to isolate, clone and produce DNA
for all the genes in appropriate quantity so that they can be sequenced
and characterized. Similarly, some of the genes which are expressed at
very low level, can be cloned and desired amount of recombinant
proteins can be produced.

2. Five steps of cloning:
1.Cutting the DNA to be cloned from the chromosomal using
sequence specific Restriction Endonuclease.
2.Selecting a cloning vector (a small molecule capable of self-
replicating inside host cells), and cutting the cloning vector with the
same restriction endonuclease producing the cohessive ends.
3.Incubating the vector and subject DNA togather to aneal and then
joining them using DNA ligase. The resultant DNA is called
recombinant DNA.
4.Transferring the reconbinant DNA to an appropriate host such as
bacteria, virus or yeast which will provide necessory biomachinary
for DNA replication.
5.Identifying the host cells that contain the recombinant DNA.

6. Cloning Vectors:
Circular plasmid DNAs are the most common cloning vectors. These
are 1 to 200 kb long DNA duplexes containing required genetic
machinery such as replication origin to permit their autonomous
propagation in host cell.
The plasmid vectors contain some specific genes responsible for
antibiotic resistance, which can be used to select the bacterial colonies
containing recombinant plasmids.
In order to clone the foreign DNA at specific site, a synthetic
oligonucleotide containing restriction sites for several REs is ligated in
the plasmid. This region is reffered as polylinker region.
Bacteriophage-based cloning vectors
Yeast artificial chromosomes vectors

8. Joining of two DNA fragments:
Dale Kaiser and Paul Berg: Used terminal deoxynucleotidyl
transferase (TdT or terminal transferase) to generate sticky or
cohesive ends in the DNA.
TdT is a mammalian enzyme which adds nucleotide to the 3’-OH
group of DNA without any requirement of primer.
The two DNA fragments to be joined, are subjected to TdT reaction in
the presence of dTTP and dATP separately to add poly-T to one DNA
and poly-A to other DNA.
The two DNA fragments with cohesive ends are annealed, the gaps are
filled with DNA polymerase and then they are joined covalently by
ligase.
TdT requires at least three nucleotides free at 3’ end (I.e unpaired), it
can be created by bacteriophage lambda exonuclease.

10. A constructed E. Coli
plasmid pBR322 designed
specially for cloning in E.
Coli.
A foreign gene cloned in
PstI restriction site can be
selected as depicted in
next slide.

11. Selection of the bacterial
colony containing recombinant
DNA by antibiotic resistance
and sensitivity.

12. Bacteriophage  cloning
vector: This virus is very
efficient in delivering its 48kb
long DNA into a host
bacterium.
One third of its DNA is non-
essential and can be replaced
by foreign DNA.
The recombinant DNA can be
packaged into phage particle
by adding this DNA to
bacterial extract containing
proteins for packaging.

13. Construction of complementary
DNA (cDNA) library:
cDNAs are the DNA with
complementary sequence to
mRNA. The cDNA represents
genes expressed at mRNA level.
1.mRNA is isolated using oligi-dT
column and annealed with oligo-
dT primer.
2.cDNA is generated by reverse
transcriptase and dNTPs
3.The mRNA is degraded by
alkaline hydrolysis and a double
standed DNA is prepared using
DNA polymerase-I and dNTPs.
4.The cDNAs created in this way
are cloned in appropriate
plasmid or phage vector and
tranfected to host cells.

14. Amplification of a DNA segment by
Polymerize Chain Reaction
(PCR):
1.DNA strands are separated by
heating.
2.Cool the DNA and add synthetic
oligonucleotide primers that flank
the region to be amplified.
3.Add thermostable DNA
polymerase (Taq1 polymerase) to
catalyse 5’-3’ synthesis of DNA.
4.Repeat steps 1, 2 and 3 30 to 40
times to generate thousands to
millions of copies of the original
DNA.

15. Colony –hybridization to screen the
bacterial plasmid library:
1.The cDNA library (bacteria
containing different cDNAs) is
plated on agar plates in appropriate
media.
2.A nitrocellulose paper is pressed
upon the the bacterial colonies.
Some bacteria are transferred to NC
paper.
3.The NC is treated with alkali to lyse
the cells and expose the cDNAs.
4.The DNA binds to NC paper, and a
radioactive DNA probe
corresponding to the desired gene is
used to hybridize with the NC
paper.
5.DNA from the Colonies with the
desired gene will be seen on X-ray
film after the exposure of the
hybridized NC paper.

16. DNA Microarray analysis for
gene expression:
1.DNA chips conatining spots
with the DNA of known
genes are available comme-
rcially, or one can make one
with desired DNAs.
2.mRNA is isolated from
control and diseased tissue
and cDNA is made using
different fluorescent
nucleotide for the two
mRNAs.
3.The fluorescent cDNAs is
then used for hybridization
with the DNA microarray
chip.
4.The fluorescent spots
indicate the expression of
corresponding gene.

17. Site-directed Mutagenesis:
Michael Smith (Canada) was awarded
with noble prize for his work on site
directed mutagenesis.
1.One can synthesize a mutated DNA
and insert into the gene using
restriction enzyme and ligase.
2.The most poular proceedure involves
working with single stranded DNA.
An oligonucleotide is made with
desired single nucleotide change and
then used as a primer with DNA
polymerase to make a mutant copy of
genes.

18. Creation of recombinant plant
using a plant parasite
agrobacterium and two
plasmid stretegy.

19. A tobacco plant in which the gene for fire fly luciferase is expressed: the
plant glows when watered with luciferin (a substrate for this enzyme).

20. Tomato plants engineered to be resistant to some insect larvae