“The technique in which a DNA fragment carrying a gene inserted into the cloning vector, this vector is introduced in the living cell and subsequent propagation of this recombination DNA molecule into many copies is known as gene cloning.

2. Gene Cloning
Presented by: Sana Farooq

3. Gene Cloning:
• “The technique in which a DNA fragment c
arrying a gene inserted into the cloning ve
ctor, this vector is introduced in the living c
ell and subsequent propagation of this rec
ombination DNA molecule into many copie
s is known as gene cloning.”

5. Gene Cloning Steps:
• There are following 5 steps that are involv
ed in gene cloning.

6. Step 1:
• A fragment of DNA, containing the gene to
be cloned, is inserted into a circular DNA
molecule called a vector, to produce a rec
ombinant DNA molecule.

7. Step 2:
• Vector transports the gene into a host cell
which is a bacterium or living cell.

8. Step 3:
• Within the host cell the vector multiplies pr
oducing numerous identical copies, not onl
y of itself but also of the gene that it carrie
s.

9. Step 4:
• When the host cell divides, copies of reco
mbinant DNA molecule are passed to the
progeny and further vector replication take
s place.

10. Step 5:
• After a large number of cell divisions, the g
enes carried by the recombinant molecule
is now said to be cloned.

13. Cloning vector
• A circular DNA molecule capable of
replication in a host cell into which a gene is
inserted to construct a recombinant DNA
molecule
• Has features that make it easier to insert
DNA and select for presence of vector in cell.
Origin of replication
Antibiotic resistance gene
Cloning site

14. Selection of vector:
1- It must be able to replicate within the host
cell
2- It must be relatively small, less than 10 kb
.

15. Plasmid
• Plasmids are circular molecules of DNA th
at lead an independent existence in bacter
ial cell and always carry one or more gene
s, and often these genes are responsible f
or a useful characteristic that gains to the
bacteria advantage.

16. • For example, the ability to survive in norm
ally toxic concentrations of antibiotics such
as chloramphenicol or ampicillin is often d
ue to the presence in the bacterium of a pl
asmid carrying antibiotic resistance genes.
• This is used as a selectable marker in cul
ture.

17. Plasmid
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nvdbvcbvb

18. Plasmid as cloning vector
• Plasmids are naturally occurring extra chro
mosomal double-stranded circular DNA m
olecules that carry
origin of replication site
a specific antibiotic resistance gene
multiple cloning site which has a number o
f unique target sites for restriction endonuc
leases.

19. Plasmid size
• Plasmid size must be less than 10 kb.
• Plasmids range from 1kb for smallest to ov
er 250kb for largest plasmid.

20. Copy number
• No. of molecules of an individual plasmid that ar
e found in a single bacterial cell.
• Types of plasmid on basis of copy number
Stringent plasmid
low copy number as 1 or 2 plasmid
Relaxed plasmid
multiple copy number as 50 or more plasmid p
er cell

21. Conjugation
• Two groups of plasmid
Conjugative plasmid
ability to sexual conjugation between ba
cterial cell for spreading plasmid and plas
mid transfer is controlled by tra genes.
Non conjugative plasmid
not able to conjugate.

22. Conjugation

23. Compatibility
• Several different kinds of plasmid may be f
ound in a single cell, including more than o
ne different conjugative plasmid at any on
e time.
• Cells of E. coli have been known to contai
n up to seven different plasmids at once.

24. • To be able to coexist in the same cell, diff
erent plasmids must be compatible.
• If two plasmids are incompatible then one
or the other will be rapidly lost from the cell
.
• Different types of plasmid can there- fore b
e assigned to different incompatibility gr
oups on the basis of whether or not they c
an coexist

25. Plasmid classification:
• Plasmids fall into five different categories:
1. Fertility plasmids-allow bacteria to mate
to each other.
2. Resistance plasmids-confer resistance t
o antibiotics or toxins.

26. 3. Degradative plasmids -enable the digest
ion of unusual substances.(toluene & salic
ylic acid)
4. Col-plasmids-encode colicins which are
proteins that kill other bacteria.
5. Virulence plasmids -turn a bacterium int
o a pathogenic strain.

27. Plasmid in eukaryotic organism
• Eukaryotic plasmid
• Is found in many strains of
Saccharomyces cerevisiae
• Has 2um size

28. Bacteriophage phage
• Viruses that infect bacteria
• Simple in structure
• Consisting of DNA or RNA that carry gene
s for replication of the phage and protectiv
e coat or capsid.

30. • Attachment and i
nsertion of phag
e DNA
• DNA replication
• Assembly and re
lease of new ph
ages
Bacterial infection by phage

31. λ phage
• An example of head and tail phage.
• λ DNA molecule is 49 kb in size and has two
features:
• Functionally related gene are clustered
together in genome as it allow genes to be
switch on and off as group
• λ DNA is found in linearized and circular form.
• Linear DNA consist of two complementary
DNA strands but at either ends it contains a
short

32. single stranded stretch of 12 nucleotides. These
stretches, are complementary and base pair
with one another to form a circular completely
double stranded structure DNA molecule, are
called sticky ends or λ cohesive ends or cos sites.
Catenane is formed in result of rolling circle
replication for production of new λ phages.
Function of cos sites is to act as recognition
sequence for endonucleases that cleaves the
catenane at cos sites, producing individual λ
genome.

33. λ
Lambda
phage
as
cloning
vector

34. M13 phage
• M13 phage is an example of filamentous type.
Its DNA has 6407 nucleotides
• It has circular but single stranded DNA.
• When it inject its DNA into the bcterial cell by
pilus, single stranded DNA molecule replicates
the DNA to form circular doulble stranded
struture. By rolling circle mechanism, ssDNA is
produced for new phages. Phage particles
assembled to form a new M13 phage.

35. M13
phage
as
cloning
vector

36. YAC vector
• Yeast artificial chromosome; eukaryotic ch
romosome require three kind of DNA sequ
ences:
Origin of DNA replication
Two telomeres to allow periodic extension
of shrinking ends by telomerase
A centeromere to ensure proper attachme
nt, via a kinetochore, to spindle microtubul
es during cell division.

37. • YAC is used to clone larger DNA fragment
s of more than 1Mb.
• Entire human gene as cystic fibrosis which
is 250kb in size.
• Survive in yeast cell.

38. Cosmid vectors
• Novel vectors that combine features of pla
smid and λ phages cosmids with cos sites.
• Larger fragments upto 45kb.

39. Cosmid vector

40. Shuttle vectors
• Certain vectors can re
plicate in different host
systems, as in E.coli a
nd in yeast. Such vect
ors are called shuttle v
ectors.
• Yep are shuttle vector
s. They replicate in E.c
oli.

41. Expression vectors
For expression the gene m
ust have a promoter, termin
ator, ribosome binding site
and other regulatory signal
recognized by bacteria.

42. Conti……
• Bacterial RNA polymerase cannot recognize
eukaryotic promoter.
• So vectors are designed in such a way that
inserted foreign gene is placed under control
of E.coli expression signals.
• Hybrid protein is obtained containig starting
few amino acid of prokaryotes and remaining
are of eukaryotes

43. Ti plasmid
• A type of plasmid that is carried by the bac
terium Agrobacterium tumefacians.
• Ti plasmid is isolated from the bacterium a
nd a DNA fragment is inserted into restricti
on site located in the T DNA region of plas
mid.

44. Ti plasmid

45. Application:
• Medicinal application
• Different genes for treatment of diseases like cys
tic fibrosis can be synthesized.
• Gene cloning play important role in production of
 Vitamin
 antibiotics

46. Growth hormone production

47. • Gene therapy
Replacement of defective genes with new
and healthy gene as to treat leukemia and
sickle cell anemia disease gene therapy is
done

48. • In research lab, cloning is used to built artif
icial, recombinant version of genes to stud
y functioning of normal genes
• Transgenic animal can be produced e.g. D
olly.