contains gene cloning & rDNA technology

1. Gene cloning and cloning
vectors
Presented by:
Syed Kashif
Department of Pharmacology
AACP
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2. DNA CLONING
 Cloning is the process of producing similar populations of genetically identical
individuals that occurs in nature.
 Cloning refers to processes used to create copies of DNA fragments (molecular
cloning), cells (cell cloning), or organisms.
 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.
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3.  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
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4.  Gene of interest is cut out with RE
 Host plasmid is cut with same RE
 Gene is inserted into plasmid and ligated with ligase
 New plasmid inserted into bacterium (transform)
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5. Enzymes used in molecular cloning
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6. Nucleases -
 Nucleases are a group of enzymes which cleave or cut the genetic material (DNA or
RNA).
 DNase and RNase Nucleases are further classified into two types based upon the
substrate on which they act. Nucleases which act on or cut the DNA are classified
as DNases, whereas those which act on the RNA are called as RNases.
 DNases are further classified into two types based upon the position where they
act. DNases that act on the ends or terminal regions of DNA are called as
exonucleases and those that act at a non-specific region in the centre of the DNA
are called as endonucleases.
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7. Restriction Enzymes
 DNAses which act on specific positions or sequences on the DNA are called as
restriction endonucleases.
 The sequences which are recognized by the restriction endonucleases or
restriction enzymes (RE) are called as recognition sequences or restriction sites.
These sequences are palindromic sequences.
 Different restriction enzymes present in different bacteria can recognize different or
same restriction sites. But they will cut at two different points within the restriction
site. Such restriction enzymes are called as isoschizomers.
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8. Mode of action
 The restriction enzyme binds to the recognition site and checks for the methylation
(presence of methyl group on the DNA at a specific nucleotide).
 If there is methylation in the recognition sequence, then, it just falls off the DNA
and does not cut. If only one strand in the DNA molecule is methylated in the
recognition sequence and the other strand is not methylated, then RE (only type I
and type III) will methylate the other strand at the required position. The methyl
group is taken by the RE from S-adenosyl methionine by using modification site
present in the restriction enzymes.
 However, type II restriction enzymes take the help of another enzyme called
methylase, and methylate the DNA. Then RE clears the DNA. If there is no
methylation on both the strands of DNA, then RE cleaves the DNA
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9. Types
 The restriction endonucleases can be divided into three groups as type I, II and III.
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10. Type I Restriction Enzymes
 These restriction enzymes recognize the recognition site, but cleave the DNA
somewhere between 400 base pairs (bp) to 10,000 bp or 10 kbp right or left.
 The cleavage site is not specific. These enzymes are made up of three peptides
with multiple functions. These enzymes require Mg++, ATP and S adenosyl
methionine for cleavage or for enzymatic hydrolysis of DNA.
 These enzymes are studied for general interest rather than as useful tools for
genetic engineering.
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11. Type II Restriction Enzymes
 Restriction enzymes of this type recognize the restriction site and cleave the DNA
withinthe recognition site or sequence.
 These enzymes require Mg++ as cofactor for cleavage activity and can generate 5
-PO4 or 3 -OH. Enzymes of this type are highly important because of their
specificity.
 Type II restriction enzymes are further divided into two types based upon their
mode of cutting.
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12. Blend End Cutters & Cohesive End Cutters
 Type II Restriction Enzymes - Blunt end cutters Type II restriction enzymes of this
class cut the DNA strands at same points on both the strands of DNA within the
recognition sequence. The DNA strands generated are completely base paired.
Such fragments are called as blunt ended or flush ended fragments.
 Type II Restriction Enzymes - Cohesive end cutter Type II restriction enzymes of this
class cut the DNA stands at different points on both the strands of DNA within the
recognition sequence. They generate a short single-stranded sequence at the end.
This short single strand sequence is called as sticky or cohesive end. This cohesive
end may contain 5 -PO4 or 3 -OH, based upon the terminal molecule (5 -PO4 or 3 -
OH). These enzymes are further classified as 5end cutter (if 5 -PO 4 is present) or 3
-end cutter (if3' -OH is present).
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13. Type III Restriction Enzymes
 Type III Restriction enzymes of this type recognize the recognition site, but cut the
DNA 1 kbp away from the restriction site. These enzymes are made up of two
peptides or subunits. These enzymes require A TP, Mg++ and S-adenosyl
methionine for action.
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14. Property Type I Type II Type III
Structure Enzyme complex of
500600 k dal
composed of three
separate subunits
Normally
homodimers of 20-
70 k dal
Heterodimers with
subunits of 70 and
100 k dal
Composition Multienzyme
complex with R
(endonuclease), M
(methylase) and S
(specificity) subunits
Separate enzymes;
endonuclease is a
homodimer,
methylase a
monomer
M subunit provides
specificity on its own;
functions as
methylase; as
heterodimer with R
subunit; functions as
methylase-
endonuclease
Cofactors Mg2+, ATP,
Sadenosylmethionine
(SAM) (needed for
cleavage as well as
methylation)
Mg2+, SAM (for
methylation only)
Mg2+, ATP (for
cleavage), SAM
(needed for
methylation:
stimulate cleavage)
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15. Property Type I Type II Type III
Recognition sites Asymmetric,
bipartite, may be
degenerate; 1315
base pairs
containing
interruption of 6 to
8 base pairs
Asymmetric, may be
bipartite, may be
degenerate; 4 to 8
base pairs normally
180° rotational
symmetry
Asymmetric,
uninterrupted, 5-6
nucleotide long with
no rotational
symmetry
Cleavage Non-specific,
variable distance
(100-1000
nucleotides) from
recognition site
Precise cleavage
within recognition
site at defined
distance
Precise cleavage at
a fixed distance; 25-
27 nucleotides from
recognition site
Example EcoK EcoRI EcoP1
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16. DNA Ligase
 Recombinant DNA experiments require the joining of two different DNA segments
or fragments in vitro.
 The cohesive ends generated by some RE will anneal themselves by forming
hydrogen bonds. But the segments annealed thus are weak and do not withstand
experimental conditions.
 To get a stable joining, the DNA should be joined by using an enzyme called ligase.
 DNA ligase joins the DNA molecule covalently by catalysing the formation of
phosphodiester bonds between adjacent nucleotides.
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17.  DNA ligase isolated from E. coli and T 4 bacteriophage is widely used.
 These ligases more or less catalyse the reaction in the same way and differ only in
requirements of cofactor.
 T4 ligase requires ATP as cofactor and E. coli ligase requires NADP as cofactor.
 The cofactor is first split (ATP - AMP + 2Pi) and then AMP binds to the enzyme to
form the enzyme-AMP complex.
 This complex then binds to the nick or breaks (with 5' -PO4 and 3' -OH) and makes
a covalent bond in the phosphodiester chain.
 The ligase reaction is carried out at 400 C for better results.
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18. Kinases
 Kinase is the group of enzymes, which adds a free pyrophosphate (PO4) to a wide
variety of substrates like proteins, DNA and RNA.
 It uses ATP as cofactor and adds a phosphate by breaking the ATP into ADP and
pyrophosphate.
 It is widely used in molecular biology and genetic engineering to add radiolabelled
phosphates.
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19. Alkaline phosphatases
 Phosphatases are a group of enzymes which remove a phosphate from a variety of
substrates like DNA, RNA and proteins.
 Phosphatases which act in basic buffers with pH 8 or 9 are called as alkaline
phosphatases.
 Most commonly bacterial alkaline phosphatases (BAP), calf intestine alkaline
phosphatases (CIAP) and shrimp alkaline phosphatases are used in molecular
cloning experiments.
 The PO4 from the substrate is removed by forming phosphorylated serine
intermediate. Alkaline phosphatase is metalloenzymes and has Zn++ ions in them.
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20. Reverse Transcriptase
 This enzyme uses an RNA molecule as template and synthesizes a DNA strand
complementary to the RNA molecule.
 These enzymes are used to synthesize the DNA from RNA.
 These enzymes are present in most of the RNA tumour viruses and retroviruses.
 Reverse transcriptase enzyme is also called as RNA dependent DNA polymerase.
 Reverse transcriptase enzyme, after synthesizing the complementary strand at the 3
end of the DNA strand, adds a small extra nucleotide stretch without
complementary sequence. This short stretch is called as R-loop.
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21. GENERATING DNA FRAGMENTS
MECHANICAL SHEARING
RESTRICTION DIGEST
CDNA SYNTHESIS
HYBRIDIZATION METHODS
DIRECT CHEMICAL SYNTHESIS

22. MECHANICAL SHEARING :
1. Random fragments of source DNA can be obtained by
mechanical shearing of bacterial, plant or animal cells.
2. Mechanical shearing caused by high speed mixing at
1500 rpm for 30 min. This gives fragments of 8 kb mean
size
3. Short single stranded regions – termini or blunt end
fragments are formed
4. Sonication can reduce the length of fragment to about
300 nucleotide pairs.
5. Shearing does not necessarily produce 5’ phosphate and
3’ OH ends. Therefore the end of the fragments must be
repaired

23. RESTRICTION ENDONUCLEASE DIGESTION :
1. Large number of restriction enzymes which recognize
and cut DNA within target sites of 4 or 5 or 6 or 7
nucleotides are known.
2. Depending upon the number of target sites present,
DNA may be cut into too small or too big size
fragments.
3. Generally the same restriction enzymes is used for
vector and the DNA of interest.
4. The digestion carried out may be light, moderate or
heavy producing from small number to large number of
fragments which are reproducible

24. PLASMID CLONING STRATEGY
It involves five steps
I. Enzyme restriction digest of DNA sample.
II. Enzyme restriction digest of DNA plasmid vector.
III. Ligation of DNA sample products and plasmid vector.
IV. Transformation with the ligation products.
V. Growth on agar plates with selection for antibiotic resistance.
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25. STEP 1. RE DIGESTION OF DNA SAMPLE25

26. STEP 2. RE DIGESTION OF PLASMID DNA26

27. STEP 3. LIGATION OF DNA SAMPLE AND PLASMID
DNA
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28. 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.
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29. CHEMICAL TRANSFORMATION WITH
CALCIUM CHLORIDE
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30. TRANSFORMATION BY ELECTROPORATION30

31. STEP 5. GROWTH ON AGAR PLATES31

32. 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
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36. CLONING VECTORS
 A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of
a higher organism, that can be stably maintained in an organism, and into which a
foreign DNA fragment can be inserted for cloning purposes.
 Cloning vectors are DNA molecules that are used to "transport" cloned sequences
between biological hosts and the test tube.
 Cloning vectors share four common properties:
 1. It should be able to replicate autonomously.
2. Contain a genetic marker (usually dominant) for selection.
3. Unique restriction sites to facilitate cloning of insert DNA.
4. Minimum amount of nonessential
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37. Types
Plasmid as vector .
Bacteriophage as vector.
Cosmid as vector
Phagemid as vector.
Bacterial Artificial Chromosomes(BACs)
Yeast Artificial Chromosomes(YACs)
Retroviral vectors
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38. Plasmid
 Bacterial cells may contain extra-chromosomal DNA called
plasmids.
 Plasmids are usually represented by small, circular DNA.
 Some plasmids are present in multiple copies in the cell
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39.  Plasmid vectors are ≈1.2–3kb and contain:
 replication origin (ORI) sequence
 a gene that permits selection,
 Here the selective gene is ampr; it encodes the enzyme b-lactamase, which
inactivates ampicillin.
 Exogenous DNA can be inserted into the bracketed region .
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40. SELECTIVE MARKER
 Selective marker is required for maintenance of plasmid in the cell.
 Because of the presence of the selective marker the plasmid becomes useful for the
cell.
 Under the selective conditions, only cells that contain plasmids with selectable marker
can survive
 Genes that confer resistance to various antibiotics are used.
 Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used
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41. ORIGIN OF REPLICATION
 Origin of replication is a DNA segment recognized by the cellular DNA-replication
enzymes.
 Without replication origin, DNA cannot be replicated in the cell.
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42. MULTIPLE CLONING SITE
 Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment
with several unique sites for restriction endo- nucleases located next to each other
 Restriction sites of the polylinker are not present anywhere else in the plasmid.
 Cutting plasmids with one of the restriction enzymes that recognize a site in the
polylinker does not disrupt any of the essential features of the vector
EcoRI – Escherichia coli strain R
BamHI – Bacillus amyloliquefaciens
DpnI – Diplococcus pneumoniae,
HindIII – Haemophilus influenzae,
BglII – Bacillus globigii,
PstI – Providencia stuartii 164,
Sau3AI – Staphylococcus aureus 3A,
KpnI – Klebsiella pneumoniae, 1st
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43. Plasmid Vector: pBR322
 Derived from E. coli plasmid ColE1), which is 4,362 bp DNA and was
derived by several alterations in earlier cloning vectors.
 pBR322 is named after Bolivar and Rodriguez, who prepared this vector.
 It has genes for resistance against two antibiotics (tetracycline and
ampicillin), an origin of replication and a variety of restriction sites for
cloning of restriction fragments obtained through cleavage with a
specific restriction enzyme.
 It has unique restriction sites for 20 restriction endonucleases.
 Certain restriction sites for eg., BAM HI in the tetr genes of the plasmid
are present within the gene in such a way that the insertion of foreign
segment of DNA will inactivate the tetr gene.
 The recombinant plasmid will allow the cells to grow only in the
presence of ampicillin but will not protect them against tetracyclin.
 Thus recombinant plasmids selection will be easily carried out.
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44. Plasmid Vector: pBR322
• Contains:
• Selectable Markers:
Ampicillin resistance gene.
Tetracycline resistance gene.
Col E I replication origin.
Eco RI site.
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45. Plasmid Vector:
 Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial
preparation I.e. University of California).
 These plasmids are 2,700 bp long and possess
 Ampicillin resistance gene
 The origin of replication derived from pBR322 and
 The lacz gene derived from E.coli.
 Within the lac region is also found a polylinker sequence having unique restriction sites.
 When DNA fragments are cloned in this region of pUC, the lac gene is inactivated.
 These plasmids when transformed into an appropriate E. coli strain, which is lac (JM103, JM109), and grown in
the presence of IPTG (isopropyl thiogalactosidase, which behaves like lactose, and induced the synthesis of b-
galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.
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46.  On the other hand, pUC having no inserts are transformed into bacteria, it will
have active lac Z gene and therefore will produce blue colonies, thus permitting
identification of colonies having pUC vector with cloned DNA segments.
 The cloning vectors belonging to pUC family are available in pairs with reverse
orders of restriction sites relative to lac Z promoter. pUC8 and pUC9 are one such
pairs.
 Other similar pairs include
 pUC12 and pUC13 and
 pUC18 and pUC19.
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47. Hind II
PstI
Sal I
BamHI
Sma I
EcoRI
EcoRI
Sma I
BamHI
Sal I
PstI
Hind II
B-galactosidase gene
pUC
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48. Plasmid Vector:
Contains
Ampicillin resistance gene.
Multiple cloning site.
ColEI ( origin ).
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49. Bacteriophage lambda (λ)
 Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as
host.
 Its structure is that of a typical phage: head, tail, tail fibres.
The bacteriophages used for cloning are the phage λ and M13 phage. It infects
bacteria.
Follow either lytic or lysogenic cycle.
There are two kinds of λ phage vectors –
insertion vector and replacement vector.
Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11
kb may be inserted.
In replacement vectors, the cleavage sites flank a region containing genes not essential
for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the
cloning process, and a larger sized DNA of 8–24 kb may be inserted.
Size is 48,502 bp.
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50. 
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51. PHAGE M13 VECTORBacteriophage lambda
COS site: Cohesive
“sticky” ends
Lysis
Lysogeny
Head
Tail
Replication
Circularized
lambda
ori
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52. Cosmids
 A cosmid is a type of hybrid plasmid that contains a Lambda phage cos
sequence.
 Cosmids are plasmids that incorporate a segment of bacteriophage λ DNA that
has the cohesive end site (cos) which contains elements required for packaging
DNA into λ particles. It is normally used to clone large DNA fragments between
28 to 45 Kb.
 Cosmid can replicate in bacterial cell, so infected cells grow into normal
colonies
 Insert DNA limited by the amount of DNA that can fit into phage capsule
 Somewhat unstable, difficult to maintain
cos
TetR
EcoRI
21.5 kb
ori
Cos site is the only
requirement for
packaging into
phage particle
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53. Latest Generation VECTOR: Phagemid- pBluescript
 A phagemid or phasmid is a plasmid that contains an f1 origin of replication from
a f1 phage.
 It can be used as a type of cloning vector in combination with filamentous phage M13.
 Phagemids contain an origin of replication (ori) for double stranded replication, as
well as an f1 ori to enable single stranded replication and packaging into phage
particles.
 Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be
introduced into a bacterial host by a range of techniques, such as
transformation and electroporation.
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55. Bacterial Artificial Chromosomes(BACs)
 BACs can hold up to 300 kbs.
 The F factor of E.coli is capable of handling large segments of DNA.
 Recombinant BACs are introduced into E.coli by electroportation ( a brief high-voltage
current). Once in the cell, the rBAC replicates like an F factor.
 Example: pBAC108L
 Has a set of regulatory genes, OriS, and repE which control F-factor replication, and
parA and parB which limit the number of copies to one or two.
 A chloramphenicol resistance gene, and a cloning segment.
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56. Yeast Artificial Chromosomes
Purpose:
Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes
Clone very large inserts of DNA: 100 kb - 10 Mb
Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome
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57.  Additional features:
 Often have a selection for an insert
 YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a selection
marker for propogation of the YAC through bacteria.
 The YAC can use both yeast and bacteria as a host
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58. RETROVIRAL VECTORS
 Retroviral vectors are used to introduce new or altered genes into the genomes of
human and animal cells.
 Retroviruses are RNA viruses.
 The viral RNA is converted into DNA by the viral reverse transcriptase and then is
efficiently integrated into the host genome
 Any foreign or mutated host gene introduced into the retroviral genome will be
integrated into the host chromosome and can reside there practically indefinitely.
 Retroviral vectors are widely used to study oncogenes and other human genes.
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59. APPLICATIONS OF GENE CLONING IN
RESEARCH
1. Identifying the genes in a genome sequence
2. Determining the function of an unknown gene
3. To study the transcriptome and proteome
I. The transcriptome, which is the messenger RNA (mRNA)
content of a cell, and which reflects the overall pattern of
gene expression in that cell.
II. The proteome, which is the protein content of a cell and
which reflects its biochemical capability.
4. Studying protein–protein interactions

60. APPLICATIONS OF GENE CLONING IN
BIOTECHNOLOGY
 Production of Protein from Cloned Genes
 Production of recombinant pharmaceuticals in medicine.
Eg. Recombinant insulin
 Synthesis of human growth hormones in E. coli
 Recombinant vaccines eg. Vaccine for hepatitis B
 The gene addition approach to plant genetic Engineering
in agiculture field eg. Plants that make their own
insecticides, Herbicide resistant crops

61. REFERENCE
Gene cloning by T.A.BROWN
Molecular Biology Of The Cell By ALBERTS
INTERNET source

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