r dna technology biochemistry

1. Recombinant
DNA technology
By dr Utsav Parmar

2. When a gene of one species is transferred to
another living organism by artificial means, it
is called recombinant DNA technology.
Also k/a genetic engineering.
Recombinant DNA technology works by
taking DNA from two different sources and
combining that DNA into a single molecule.

3.  It occurs in following stages
 Generation of DNA fragments & selection of
the desired piece of DNA (e.g. a human
gene).
 Insertion of the selected DNA into a cloning
vector (e.g. a plasmid) to create a
recombinant DNA or chimeric DNA.

4.  Introduction of the recombinant vectors
into
host cells (e.g. bacteria).
 Multiplication & selection of clones
containing the recombinant molecules.
 Expression of the gene to produce the
desired product.

5. Five Basic Steps in Gene Cloning
1. choose the appropriate DNA to be cloned,
genomic or cDNA.
2. Produce a collection of DNA fragments of
size suitable for inserting into appropriate
vectors.
3. Insert DNA fragments into the vector using
DNA ligase (DNA ligation.)
4. Introduce DNA fragments into a population
of bacteria (transformation.)
5. Select the colonies containing desired
sequence from the “library.”

8. Isolation of target DNA

9. TOOLS OF RECOMBINANT DNA
TECHNOLOGY
• Enymes
• Vectors
• Hosts
• DNA to be cloned

10. ENZYMES
• Nucleases
• Exonucleases
• Endonucleases
• Restriction endonucleases

11. Restriction endonucleases
 DNA cutting enzymes:
 Restriction endonucleases are one of the
most important groups of enzymes for the
manipulation of DNA.
 These are the bacterial enzymes that can
cut/split DNA (from any source) at specific sites.

12. • They were first discovered in E.coli restricting
the replication of bacteriophages (virus), by
cutting the viral DNA (The host E. coli DNA is
protected from cleavage by addition of
methyl groups).
 Thus, the enzymes that restrict the viral
replication are known as restriction enzymes
or restriction endonucleases.

13.  Recognition sequence is the site where the DNA is
cut by a restriction endonuclease.
 Restriction endonucleases can specifically
recognize DNA with a particular sequence of 4-8
nucleotides & cleave.
 Cleavage patterns: Majority of restriction
endonucleases (particularly type II) cut DNA
at defined sites within recognition sequence.

14.  The same sequence of bases is found on
both DNA strands, but in opposite order.
 This arrangement is called a palindrome.
Palindromes are words or sentences that
read the same forward and backward.

15.  The cut DNA fragments by restriction
endonucleases may have mostly sticky ends
(cohesive ends) or blunt ends.
 DNA fragments with sticky ends are useful for
recombinant DNA experiments.
 This is because the single-stranded sticky
DNA
ends can easily pair with any other DNA
fragment having complementary sticky ends.

19.  The cut DNA fragments are covalently
joined
together by DNA ligases.
 These enzymes were originally isolated from
viruses.
 They also occur in E.coli & eukaryotic cells.
 DNA ligases actively participate in cellular
DNA repair process.

20.  The hosts are the living systems or cells in
which the carrier of recombinant DNA
molecule or vector can be propagated.
 There are different types of host cells-
prokaryotic (bacteria) & eukaryotic
(fungi, animals & plants).

21.  Host cells, besides effectively
incorporating the vector's genetic
material, must be conveniently cultivated
in the laboratory to collect the products.
 Microorganisms are preferred as host
cells, since they multiply faster compared
to cells of higher organisms (plants or
animals).

22.  Escherichia coli:
 Escherichia coli was the first organism used
in the DNA technology & continues to be
the host of choice by many workers.
 The major drawback is that E. coli (or even
other prokaryotic organisms) cannot
perform post-translational modifications.
 Bacillus subtilis as an alternative to E.coli.

23.  The most commonly used eukaryotic organism
is the yeast, Saccharomyces cerevisiae.
 Certain complex proteins which cannot be
synthesized by bacteria can be produced by
mammalian cells e.g. tissue plasminogen activator.
 The mammalian cells possess the machinery to
modify the protein to the active form (post-
translational modifications).

24.  Vectors are the DNA molecules, which
can carry a foreign DNA fragment to be
cloned.
 They are self-replicating in an appropriate
host cell.
 The most important vectors are plasmids,
bacteriophages, cosmids & artificial
chromosome vectors.

25. Ideal vector
A vector should have four characteristics:
1.Ability to replicate independently of the host cell
2.A recognition sequence for a restriction enzyme
(cloning site)
3.One or more selectable/reporter genes
4.Small size in comparison with host’s
chromosomes

26.  Plasmids are extrachromosomal,
double- stranded, circular, self-
replicating DNA molecules.
 Almost all bacteria have plasmids.
 Size of plasmids varies from 1 to 500 kb.
 Plasmids contribute to about 0.5 to 5.0%
of total DNA of bacteria.

27.  pBR322 has a DNA sequence of 4,361 bp.
 It carries genes resistance for ampicillin (Amp1) &
tetracycline (Tel1) that serve as markers for the
identification of clones carrying plasmids.
 The plasmid has unique recognition sites for the
action of restriction endonucleases - EcoRl,
Hindlll, BamHl, Sall & Pstll

29.  The other plasmids employed as cloning
vectors include pUC19 (2,686 bp, with
ampicillin resistance gene) & derivatives
of pBR322-pBR325, pBR328 & pBR329.

30.  Bacteriophages or phages are the viruses
that replicate within the bacteria.
 In case of certain phages, their DNA gets
incorporated into the bacterial chromosome
& remains there permanently.

31.  Phages can take up larger DNA segments
than plasmids.
 Phage vectors are preferred for working
with genomes of human cells.
 The most commonly used phages are
bacteriophage λ (phage λ) &
bacteriophage (phage M13).

32.  Cosmids are vectors possessing the characteristics of both
plasmid & phage λ.
 Cosmids can be constructed by adding a fragment of
phage λ DNA including Cos site, to plasmids.
 A foreign DNA (about 40 kb) can be inserted into cosmid
DNA .
 The recombinant DNA, formed can be packed as phages &
injected into E.coli.
 Inside host cell, cosmids behave like plasmids & replicate &
can carry larger fragments of foreign DNA

33.  Human artificial chromosome (HAC):
 Artificial chromosome is a synthetically
produced vector DNA, possessing the
characteristics of human chromosome.
 HAC may be considered as a self-replicating
microchromosome with a size ranging from 1/10th
to 1/5th of a human chromosome.
 It can carry long human genes.

34.  Yeast artificial chromosome (YAC) is a
synthetic DNA that can accept large
fragments of foreign DNA (particularly
human DNA).
 It is possible to clone large DNA pieces
by
using YAC.

35.  Construction of BACs is based on one F-
plasmid which is larger than the other
plasmids used as cloning vectors.
 BACs can accept DNA inserts of around 300
kb.

36.  Transformation:
 Transformation is the method of
introducing
foreign DNA into bacterial cells (e.g. E.coli).
 Uptake of plasmid DNA by E.coli is carried
out in ice-cold CaCl2 (0-5˚C) & a
subsequent
heat shock (37-45˚C for about 90 sec).

37.  A natural microbial recombination process.
 During conjugation, two live bacteria (a
donor & a recipient) come together, join by
cytoplasmic bridges & transfer single
stranded DNA (from donor to recipient).
 In side recipient cell, new DNA may
integrate
with the chromosome or may remain free.

38.  It is a technique involving electric field
mediated membrane permeabiIization.
 Electric shocks can also induce cellular
uptake of exogenous DNA (believed to be
via the pores formed by electric pulses) from
the suspending solution.
 It is a simple & rapid technique for
introducing genes into cells.

39.  Liposomes are circular lipid molecules,
which have an aqueous interior that can
carry nucleic acids.
 Several techniques have been developed
to encapsulate DNA in liposomes.
 The liposome mediated gene transfer is
referred to as lipofection.

40.  Treatment of DNA fragment with liposomes,
DNA pieces get encapsulated inside liposomes.
 These liposomes can adhere to cell
membranes
& fuse with them to transfer DNA fragments.
 The DNA enters the cell & to the nucleus.
 Positively charged liposomes efficiently
complex with DNA, bind to cells & transfer DNA

41.  It is possible to directly transfer the DNA into
the cell nucleus.
 Microinjection & particle bombardment
are the two techniques used for this
purpose.

42. Selection and screening Identification of host cells containing
recombinant DNA requires genetic selection
or screening or both.
 In a selection, cells are grown under
conditions in which only transformed cells
can survive; all the other cells die.
 In contrast, in a screen, transformed cells
have to be individually tested for the
presence of the desired recombinant DNA.
 Many selection strategies involve selectable
marker genes— genes whose presence can
easily be detected or demonstrated

44. Screening (Strategies)1. Gel Electrophoresis Allows Separation of
Vector DNA from Cloned Fragments
2. Cloned DNA Molecules Are Sequenced
Rapidly by the Dideoxy Chain-Termination
Method
3. The Polymerase Chain Reaction Amplifies
a Specific DNA Sequence from a Complex
Mixture
4. Blotting Techniques Permit Detection of
Specific DNA Fragments and mRNAs with
DNA Probes

46. 1. Analysis of Gene Structure and Expression
2. Pharmaceutical Products
 Drugs
 Vaccines
1. Genetically modified organisms (GMO)
 Transgenic plants
 Transgenic animal
1. Application in medicine
- Gene therapy

47. Analysis of Gene Structure and
Expression
 Using specialized recombinant DNA
techniques, researchers have determined
vast amounts of DNA sequence including the
entire genomic sequence of humans and
many key experimental organisms.
 This enormous volume of data, which is
growing at a rapid pace, has been stored
and organized in the GenBank at the National
Institutes of Health, Bethesda, Maryland

48. Pharmaceutical Products Some pharmaceutical applications of DNA
technology:
 Large-scale production of human hormones
and other proteins with therapeutic uses
 Production of safer vaccines
 A number of therapeutic gene products —
insulin, the interleukins, interferons, growth
hormones, erythropoietin, and coagulation
factor VIII—are now produced commercially
from cloned genes

50. Genetically modified organisms
(GMO)Use of recombinant plasmids
in agriculture
plants with genetically
desirable traits
 herbicide or pesticide resistant
corn & soybean
 Decreases chemical insecticide use
 Increases production
 “Golden rice” with beta-carotene
 Required to make vitamin A, which in
deficiency causes blindness

51.  Crops have been
developed that are
better tasting, stay
fresh longer, and are
protected from
disease and insect
infestations.
“Golden rice” has been
genetically modified to
contain beta-carotene

52. Insect-resistant tomato plants
The plant on the left contains a gene that
encodes a bacterial protein that is toxic to
certain insects that feed on tomato plants.
The plant on the right is a wild-type plant.
Only the plant on the left is able to grow
when exposed to the insects.

53. Transgenic animals
Green fluorescence Red fluorescence

54. Transgenic animals