1) Gene cloning involves inserting foreign DNA into a plasmid vector, transforming bacteria with the recombinant plasmid, and allowing the bacteria to replicate, producing multiple copies of the gene of interest. 2) Restriction enzymes are used to cut the DNA at specific sites, producing sticky ends that allow insertion of the gene into the plasmid. 3) Transformed bacteria containing the recombinant plasmid are selected by growing on antibiotic-containing media, with the antibiotic resistance gene on the plasmid allowing only those bacteria to survive.

1. Principles of cloning,
vectors and cloning
strategies
Dr/ Ahmed Abdellatif
Assistant Professor at Faculty of Pharmacy,
- Qassim University, Kingdom of Saudi Arabia.
- Al-Azhar University, Egypt.

2. DNA Cloning and Its Applications:
A Preview
 Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use
of bacteria and their plasmids
 Plasmids are small circular DNA molecules that
replicate separately from the bacterial
chromosome
 Cloned genes are useful for making copies of a
particular gene and producing a protein product
© 2011 Pearson Education, Inc.

3.  Gene cloning involves using bacteria to make
multiple copies of a gene
 Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial
cell
 Reproduction in the bacterial cell results in
cloning of the plasmid including the foreign
DNA
 This results in the production of multiple copies
of a single gene
© 2011 Pearson Education, Inc.

4. Bacterium
Bacterial
chromosome
Plasmid
2
1
3
4
Gene inserted into
plasmid
Cell containing gene
of interest
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium
Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Gene of
interest
Protein expressed from
gene of interest
Protein harvestedCopies of gene
Basic research
and various
applications
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth

5. DNA CLONING
 DNA cloning is a technique for
reproducing DNA fragments.
 It can be achieved by two different
approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
 a vector is required to carry the DNA
fragment of interest into the host cell.

6. DNA CLONING
 DNA cloning allows a copy of any specific part of
a DNA (or RNA) sequence to be selected among
many others and produced in an unlimited amount.
 This technique is the first stage of most of the
genetic engineering experiments:
▪ Production of DNA libraries
▪ PCR
▪ DNA sequencing

7. Gene Cloning
 Most methods for cloning pieces of DNA share
certain general features.
 For example, a foreign gene is inserted into a bacterial
plasmid and this recombinant DNA molecule is returned to
a bacterial cell.
 Every time this cell reproduces, the recombinant plasmid is
replicated as well and passed on to its descendants.
 Under suitable conditions, the bacterial clone will make the
protein encoded by the foreign gene.

8. DNA CLONING
 Massive amplification of DNA sequences
 Stable propagation of DNA sequences
 A single DNA molecule can be amplified
allowing it to be:
▪ Studied - Sequenced
▪ Manipulated - Mutagenized or Engineered
▪ Expressed - Generation of Protein

9.  One basic cloning technique begins with the insertion of a
foreign gene into a bacterial plasmid.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.1

10. CLONING PROCESS
 Gene of interest is cut out with
Restriction Enzymes (RE )
 Host plasmid is cut with same RE
 Gene is inserted into plasmid and
ligated with ligase
 New plasmid inserted into
bacterium (transform)

11. CLONING PROCESS

12. PLASMID CLONING STRATEGY
 Involves five steps:
Enzyme restriction digest of DNA sample.
Enzyme restriction digest of DNA plasmid vector.
Ligation of DNA sample products and plasmid vector.
Transformation with the ligation products.
Growth on agar plates with selection for antibiotic
resistance.

13. Restriction Enzymes
 In nature, bacteria use restriction
enzymes to cut foreign DNA, such as
from phages or other bacteria.
 Most restrictions enzymes are very
specific, recognizing short DNA
nucleotide sequences and cutting at
specific point in these sequences.

14.  Each restriction enzyme cleaves a specific
sequence of bases called a restriction site.
 These are often a symmetrical series of four to eight
bases on both strands running in opposite directions.
 If the restriction site on one strand is 3’-CTTAAG-5’,
the complementary strand is 5’-GAATTC-3
 Restriction enzymes cut covalent phosphodiester
bonds of both strands, often in a staggered way
creating single-stranded ends, sticky ends.
 These extensions will form hydrogen-bonded base
pairs with complementary single-stranded stretches
on other DNA molecules cut with the same restriction
enzyme.

16.  Recombinant plasmids are produced by splicing
restriction fragments from foreign DNA into
plasmids.
 A plasmid is a circular piece of DNA found in
bacteria and contain genes.
 Plasmids can be used to insert DNA from another
organism into a bacterial cell.
 Then, as a bacterium carrying a recombinant
plasmid reproduces, the plasmid replicates within it.
Recombinant DNA vectors

17. • Bacteria are most commonly used as host
cells for gene cloning because DNA can be easily
isolated and reintroduced into their cells.
• Bacteria cultures also grow quickly, rapidly
replicating the foreign genes.
•Bacteria will also produce large amounts of the
protein of interest

18.  The process of
cloning a gene in a
bacterial plasmid can
be divided into five
steps.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.3

19. STEP 1. RE DIGESTION
OF DNA SAMPLE

20. 1. Isolation of vector and gene-source DNA.
 The source DNA may come from
human tissue cells.
 The source of the plasmid is
typically E. coli.
 This plasmid carries useful genes,
such as ampR, conferring resistance
to the antibiotic ampicillin

21. STEP 2. RE DIGESTION OF
PLASMID DNA
The gene product of lacZ is β-
galactosidase which cleaves lactose, a
disaccharide, into glucose and galactose.
Plasmids are also an efficient means of
amplifying cloned DNA because there
are many copies per cell, as many as
several hundred for some plasmids.
Plasmid pBR322 is simpler in structure; it
has two drug-resistance genes, tetR and
ampR. Both genes contain unique
restriction target sites that are useful in
cloning.

22. 2. Insertion of DNA into the vector.
 By digesting both the plasmid and human DNA with
the same restriction enzyme we can create thousands
of human DNA fragments, one fragment with the
gene that we want, and with compatible sticky ends
on bacterial plasmids.
 After mixing, the human fragments and cut plasmids
form complementary pairs that are then joined by
DNA ligase.
 This creates a mixture of recombinant DNA
molecules.

23. Using Restriction Enzymes to Make
Recombinant DNA
 Bacterial restriction enzymes cut DNA molecules at
specific DNA sequences called restriction sites
 A restriction enzyme usually makes many cuts,
yielding restriction fragments
 The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky
ends.”
© 2011 Pearson Education, Inc.

24.  Sticky ends can bond with
complementary sticky ends of
other fragments
 DNA ligase is an enzyme that
seals the bonds between restriction
fragments
© 2011 Pearson Education, Inc.

25. Figure 20.3-1
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
3
3
3
3
3
3
1
Sticky
end
GAATTC
CTTAAG

26. Figure 20.3-2
One possible combination
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
5
5
55
5
5
55
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
1
Sticky
end
GAATTC
CTTAAG
G
G
G
G
AATT CAATT C
C TTAA C TTAA

27. Figure 20.3-3
Recombinant DNA molecule
One possible combination
DNA ligase
seals strands
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
5
5
55
5
5
55
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
1
Sticky
end
GAATTC
CTTAAG
G
G
G
G
AATT CAATT C
C TTAA C TTAA

28. Cloning a Eukaryotic Gene in a
Bacterial Plasmid
 In gene cloning, the original plasmid
is called a cloning vector
 A cloning vector is a DNA molecule
that can carry foreign DNA into a
host cell and replicate there
© 2011 Pearson Education, Inc.

29.  Some recombinant plasmids now contain
hummingbird DNA
 The DNA mixture is added to bacteria that have
been genetically engineered to accept it
 The bacteria are plated on a type of agar that
selects for the bacteria with recombinant
plasmids
 This results in the cloning of many
hummingbird DNA fragments, including the β-
globin gene
© 2011 Pearson Education, Inc.

30. Figure 20.4
Bacterial plasmid
TECHNIQUE
RESULTS
ampR gene lacZ gene
Restriction
site
Hummingbird cell
Sticky
ends Gene of
interest
Humming-
bird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
Bacteria carrying
plasmids
Colony carrying non-
recombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones

31. Figure 20.4a-1
Bacterial plasmid
TECHNIQUE
ampR gene lacZ gene
Restriction
site
Hummingbird cell
Sticky
ends Gene of
interest
Humming-
bird DNA
fragments

32. Figure 20.4a-2
Bacterial plasmid
TECHNIQUE
ampR gene lacZ gene
Restriction
site
Hummingbird cell
Sticky
ends Gene of
interest
Humming-
bird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid

33. Figure 20.4a-3
Bacterial plasmid
TECHNIQUE
ampR gene lacZ gene
Restriction
site
Hummingbird cell
Sticky
ends Gene of
interest
Humming-
bird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
Bacteria carrying
plasmids

34. Figure 20.4b
RESULTS
Bacteria carrying
plasmids
Colony carrying non-
recombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones

35. Storing Cloned Genes in DNA Libraries
 A genomic library that is made using
bacteria is the collection of
recombinant vector clones produced by
cloning DNA fragments from an entire
genome
 A genomic library that is made using
bacteriophages is stored as a collection
of phage clones
© 2011 Pearson Education, Inc.

36. STEP 3. LIGATION OF DNA
SAMPLE AND PLASMID DNA

37. 3. Introduction of the cloning vector into cells.
 Bacterial cells take up the recombinant
plasmids by transformation.
 This creates a diverse pool of bacteria, some
bacteria that have taken up the desired
recombinant plasmid DNA, other bacteria that
have taken up other DNA, both recombinant
and nonrecombinant.

38. STEP 4. TRANSFORMATION
OF LIGATION PRODUCTS
 The process of transferring exogenous DNA into
cells is call “transformation”
 There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to promote
DNA entry into cells.
 A second method is called electroporation based on
a short pulse of electric charge to facilitate DNA
uptake.

39. CHEMICAL TRANSFORMATION WITH
CALCIUM CHLORIDE

40. TRANSFORMATION BY
ELECTROPORATION

41. 4. Cloning of cells (and foreign genes).
 We can plate out the transformed bacteria
on a solid nutrient medium containing
ampicillin.
 Only bacteria that have the ampicillin-
resistance plasmid will grow.
5. Identifying cell clones with the right gene.
 In the final step, we will sort through the
thousands of bacterial colonies with foreign
DNA to find those containing our gene of
interest.

42.  One technique to test the clones of
interest is to use DNA or Nucleic acid
hybridization.
 This technique depends on base pairing
between our gene and a short piece of
DNA or RNA with a complementary
sequence to the gene called a Probe,
 The sequence of our RNA or DNA probe
depends on knowledge of at least part of
the sequence of our gene.
 A radioactive or fluorescent tag labels the
probe so that if it bind with our gene we
can detect it’s presence.

43. STEP 5. GROWTH ON AGAR
PLATES

44. STEP 5
 Blue colonies represent Ampicillin-resistant
bacteria that contain pVector and express a
functional alpha fragment from an intact
LacZ alpha coding sequence.
White colonies represent Ampicillin-resistant
bacteria that contain pInsert and do not
produce LacZ alpha fragment

45. TERMS USED IN CLONING
 DNA recombination.
The DNA fragment to be cloned is inserted
into a vector.
 Transformation.
The recombinant DNA enters into the host
cell and proliferates.
 Selective amplification.
A specific antibiotic is added to kill E. coli
without any protection. The transformed E.
coli is protected by the antibiotic-resistance
gene
 Isolation of desired DNA clones

46. AGAROSE GEL
ELECTROPHORESIS

48. APPLICATIONS
 Cloning DNA fragments
 Generating Libraries: essential step for
genome mapping
 Positional cloning – discovering disease
genes
 Discovering genes from e.g. Protein
sequence

49. ANALYSIS OF CLONED
DNA
 Is it the one you wanted?
 What are its molecular characteristics?

50.  Restriction mapping: determining the order
of restriction sites in a cloned fragment:
 Gel electrophoresis: separates DNA
fragments by molecular weight
 Southern Blot analysis: DNA is transferred
("blotted") to filter paper.Filter is exposed to a
DNA probe. Binds specifically to target DNA
immobilized on filter
 DNA sequencing: provides complete order
of bases in a DNA fragment

52. Gel Electrophoresis

53. Loading Gel

54. LoadedGel

55. RunGel

56. 2-DProteinElectrophoresis

57. 2-DProteinElectrophoresis Visualized proteins

58. RestrictionFragmentAnalysis No need to know
details of what
gene was studied
or specific
restriction
enzyme used

59. SouthernBlotting

60. GenomicMapping

61. DNASequencing

62. DNASequencing

63. DNASequencing

64. DNASequencer

65. ShotgunSequencing