in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.

2. A gene is a basic physical
and functional unit of
heredity. Genes which are
made up of DNA, acts as
instructions to molecules
called proteins.

3. Cloning is the process
of generating an
identical copy of a cell
or an organism. Clones
are those organisms
that have identical
genes.

4. “Gene cloning is genetic engineering
technique in which a gene of interest is
fused into a plasmid and introduced into a
suitable host, self replicates and generates a
large number of identical copies of the
particular gene.”

5. STEPS INVOLVED IN GENE CLONING:
Generation of foreign DNA molecules
Selection of suitable vector
Insertion of foreign DNA into vector
Recombinant DNA introduced into host cells
Selection and screening methods of
recombinant clones

7. Strategies to generate foreign DNA
molecules:
Using restriction enzymes
cDNA synthesis
Using gene machine

8. USING RESTRICTION ENZYMES:
 Restriction enzyme is a dna cutting enzyme
that recognizes specific sites in DNA, they are
often called as restriction endonuclease.
 It cleaves the dna in two forms. They are
i. Blunt end
ii. cohesive end

10. Eg:
Eco K1
Eco A1
Cfr A1
Eg:
Eco R1
Bam H1
Hind III
Eg:
Eco P1
Hinf III
EcoP15i

11. cDNA SYNTHESIS:
 Reverse transcription is the process of making
a double stranded DNA molecule from a single
stranded RNA template catalyzed by an
enzyme reverse transcriptase.
 it is also called as RNA-dependent DNA
polymerase.

14. USING GENE MACHINE:
“Gene machine is a fully automated
commercial instrument, also called as
automated polynucleotide synthesizer
which synthesizes predetermined
polynucleotide sequence.”

15. Working principle:
“Development of insoluble silica-based
support in the form of beads which provides
support for solid phase synthesis of DNA
chain.”
“Development of stable deoxyribonucleoside
phosphoramides as synthons which are stable
to oxidation and hydrolysis, and ideal for
DNA synthesis.”

17. vector
Vector is a extra chromosomal double
stranded circular DNA which has the ability
to self replicate.
It acts as a carrier molecule in which foreign
DNA can be inserted or integrated and
replicates in the host cell to produce multiple
clones of recombinant DNA.

19. VECTOR SIZE:
The plasmid size less
than 10 kb is desirable
for a cloning vector.
Plasmids ranges from
1.0kb to 250kb,but only
few are used for cloning.
Selection of suitable cloning vector

20. Copy number:
 The copy number refers to the number of
molecules of an individual plasmid that are
normally found in a single bacterial cell.
 Based on copy number plasmids are classified
into two types:
i. Stringent plasmids – high copy number
ii. Relaxed plasmids – low copy number

21. Selectable marker:
 The selectable marker gene permits the
selection of host cells which bear recombinant
DNA (transformants) from those which do not
bear rDNA(non-transformants).
 Two types of selectable markers
1. Drug-resistance markers
2. Auxotrophic markers

23. Restriction sites:
 Restriction sites allow cleavage of specific
sequence by specific restriction endonuclease
to insert the desired DNA fragment.
 Restriction sites in E.coli cloning vector
includes Hind III, BamH1, EcoR1, Sal1, PvuI,
CIaI etc.

24. ORI:
Origin of replication(ORI) is a specific
sequence of nucleotide in DNA is linked to
this sequence then along with vector
replication, foreign DNA also starts
replicating within host cell.