RECOMBINANT DNA RECOMBINANT DNA RECOMBINANT DNA.pdf

RECOMBINANT DNA

1. Guru Nanak Khalsa College
Yamuna Nagar 2022
RECOMBINANT DNA
TECHNOLOGY: PROCESS & APPLICATIONS
COURSE:MOLECULAR CELLBIOLOGY
CLASS&SECTION:MSC.BIOTECHNOLGY (PREVIOUSYEAR)
COURSECORDINATOR:DR.NIDHIMAHENDRU
DESIGNATION:ASSISTANTPROFESSOR
DEPARTMANT :DEPARTMENT OFBIOTECHNOLGY
PURPOSE:CLASSROOM LEARNING(CONCEPTLEARNING)

2. Recombinant DNA
Technology

5. Recombinant DNA and
Gene Cloning
Recombinant DNA(rDNA) is a form of artificial
DNA that is created by combining two or more
sequences that would not normally occur together
through the process of gene splicing.
Recombinant DNA technologyis a technology
which allows DNA to be produced via artificial
means. The procedure has been used to change
DNA in living organisms and may have even more
practical uses in the future.

6. Recombinant DNA:
Cloning and Creation of Chimeric Genes

7. Recombinant DNA technology is
one of the recent advances in
biotechnology, which was
developed by two scientists named
Boyerand Cohenin 1973.

8. What is Recombinant DNA Technology?
Recombinant DNA technologyis a
technology which allows DNA to be
produced via artificial means.
The procedure has been used to change
DNA in living organisms and may have even
more practical uses in the future.
It is an area of medical science that is just
beginningto be researched in a concerted
effort.

9. Recombinant DNA technology works by
taking DNA from two different sources and
combiningthat DNA into a single molecule.
That alone, however, will not do much.
Recombinant DNA technology only becomes
useful when that artificially-created DNA is
reproduced. This is known as DNA cloning.

10. Brief Introduction

11. Recombinant DNA Technology
1.The basic concepts for recombinant
DNA technology
2.The basic procedures of recombinant
DNA technology
3.Application of recombinant DNA
technology

12. The basic concepts for
recombinant DNA technology

13. In the early 1970s, technologies for the
laboratory manipulation of nucleic acids
emerged. In turn, these technologies led to
the construction of DNA molecules
composed of nucleotide sequences taken
from different sources. The products of
these innovations, recombinant DNA
molecules, opened exciting new avenues of
investigation in molecular biology and
genetics, and a new field was born—
recombinant DNA technology.

14. Concept of Recombinant DNA
Recombinant DNAis a molecule that combines
DNA from two sources . Also known as gene
cloning.
Creates a new combination of genetic material
–Human gene forinsulinwas placed in bacteria
–The bacteria are recombinant organisms and
produce insulin in large quantities for diabetics
–Genetically engineered drug in 1986
Genetically modified organisms are possible
because of the universal nature of the genetic
code!

15. Genetic engineeringis the application of
this technology to the manipulation of
genes. These advances were made
possible by methods for amplification of
any particular DNA segment( how? ),
regardless of source, within bacterial
host cells. Or, in the language of
recombinant DNA technology, the
cloning of virtually any DNA sequence
became feasible.

16. Recombinant technology begins with the
isolation of a gene of interest (target gene).
The target gene is then inserted into the
plasmid or phage (vector) to form replicon.
The replicon is then introduced into host cells
to cloned and either express the protein or not.
The cloned replicon is referred to as
recombinant DNA. The procedure is called
recombinant DNA technology. Cloning is
necessary to produce numerous copies of the
DNA since the initial supply is inadequate to
insert into host cells.

17. Some other termsare also in common use to
describe genetic engineering.
Gene manipulation
Recombinant DNA technology
Gene cloning (Molecular cloning)
Genetic modification

18. Cloning——In classical biology, a clone is a
population of identical organisms derived
from a single parental organism.
For example, the members of a colony of
bacterial cells that arise from a single cell on a
petri plate are clones. Molecular biology has
borrowed the term to mean a collection of
molecules or cells all identical to an original
molecule or cell.

19. How recombinant technology works
These steps include isolatingof the target
geneand the vector,specific cuttingof
DNA at defined sites, joiningor splicing of
DNA fragments, transforming of replicon
to host cell,cloning, selectingof the positive
cells containing recombinant DNA, and
either express or not in the end.

20. Six steps ofRecombinant DNA
1.Isolating (vectorand target gene)
2.Cutting (Cleavage)
3.Joining (Ligation)
4.Transforming
5.Cloning
6.Selecting (Screening)

21. Recombinant DNA Technology
1.The basic concepts for recombinant
DNA technology
2.The basic procedures of recombinant
DNA technology
3.Application of recombinant DNA
technology

22. The basic procedures of
recombinant DNA technology

23. DNA molecules that are constructed with DNA
from different sources are called recombinant
DNA molecules.
Recombinant DNA molecules are created in
nature more often than in the laboratory;
–for example, every time a bacteria phage or
eukaryotic virus infects its host cell and
integrates its DNA into the host genome, a
recombinant is created.
–Occasionally, these viruses pick up a fragment
of host DNA when they excise fromtheir host’s
genome; these naturally occurring
recombinant DNA molecules have been used to
study some genes.

24. Six basic steps are common to most
recombinant DNA experiments
1.Isolation and purificationof DNA.
Both vectorand target DNAmolecules
can be prepared by a variety of
routine methods, which are not
discussed here. In some cases, the
target DNA is synthesized in vitro.

25. 2. Cleavage of DNA at particular sequences.As
we will see, cleaving DNA to generate
fragments of defined length, or with specific
endpoints, is crucial to recombinant DNA
technology. The DNA fragment of interest is
called insert DNA. In the laboratory, DNA is
usually cleaved by treating it with
commercially produced nucleases and
restriction endonucleases.

26. 3. Ligationof DNA fragments.
A recombinant DNA molecule is usually
formed by cleaving the DNA of interest to
yield insert DNA and then ligatingthe insert
DNA to vector DNA (recombinant DNAor
chimeric DNA). DNA fragments are
typically joined using DNA ligase(also
commercially produced).
–T4 DNA Ligase

27. 4. Introduction of recombinant DNA into
compatible host cells.In order to be
propagated, the recombinant DNA
molecule (insert DNA joined to vector
DNA) must be introduced into a
compatible host cell where it can replicate.
The direct uptake of foreign DNA by a host
cell is called genetic transformation(or
transformation). Recombinant DNA can
also be packaged into virus particles and
transferred to host cells by transfection.

28. 5. Replication and expression of
recombinant DNA in host cells.
Cloning vectors allow insert DNA to be
replicated and, in some cases, expressed
in a host cell. The ability to clone and
express DNA efficiently depends on the
choice of appropriate vectors and hosts.

29. 6. Identification of host cells that contain
recombinant DNA of interest.Vectors
usually contain easily scored genetic
markers, or genes, that allow the
selection of host cells that have taken up
foreign DNA. The identification of a
particular DNA fragment usually
involves an additional step—screening a
large number of recombinant DNA
clones. This is almost always the most
difficultstep.

30. DNA cloning in a plasmid
vector permits amplification
of a DNA fragment.

31. First step:
Isolating DNA
1.Vector
2.Target gene

32. How to get a target genes?
1.Genomic DNA
2.Artificial synthesis
3.PCR amplification
4.RT-PCR

33. Polymerase chain reaction(PCR)
A technique called the polymerase chain
reaction (PCR)has revolutionized
recombinant DNA technology. It can
amplify DNA from as little material as a
single cell and from very old tissue such
as that isolated from Egyptian mummies,
a frozen mammoth, and insects trapped
in ancient amber.

34. method is used to
amplify DNA
sequences
The polymerase chain
reaction (PCR)can
quickly clone a small
sample of DNA in a
test tube
Number of DNA
molecules
Initial
DNA
segment

35. PCR primers

36. RT-PCR
Reverse transcription polymerase chain reaction
(RT-PCR) is a variant of polymerase chain
reaction (PCR.
In RT-PCR, however, an RNA strandis first
reverse transcribed into its DNA complement
(complementary DNA, or cDNA) using the enzyme
reverse transcriptase, and the resulting cDNA is
amplified using traditional.
–Template:RNA
–Products: cDNA

37. Vectors-Cloning Vehicles
Cloning vectorscan be plasmids,
bacteriophage, viruses, or even small
artificial chromosomes. Most vectors
contain sequences that allow them to be
replicated autonomouslywithin a
compatible host cell, whereas a minority
carry sequences that facilitate integration
into the host genome.

38. All cloning vectors have in common at least
one uniquecloning site, a sequence that can
be cut by a restriction endonucleaseto allow
site-specific insertion of foreign DNA. The
most useful vectors have several restriction
sites grouped together in a multiple cloning
site (MCS) called a polylinker.

39. Types of vector
1.Plasmid Vectors
2.Bacteriophage Vectors
3.Virus vectors
4.Shuttle Vectors--can replicate in either
prokaryotic or eukaryotic cells.
5.Yeast Artificial Chromosomes as
Vectors

40. Plasmid Vectors
Plasmidsare circular, double-stranded
DNA (dsDNA)molecules that are separate
from a cell’s chromosomal DNA.
These extra chromosomal DNAs, which
occur naturally in bacteria and in lower
eukaryotic cells (e.g., yeast), exist in a
parasitic or symbiotic relationship with
their host cell.

41. Plasmid

42. Plasmids can replicate autonomously within
a host, and they frequently carry genes
conferring resistance to antibiotics such as
tetracycline, ampicillin, or kanamycin. The
expression of these marker genes can be
used to distinguish between host cells that
carry the vectors and those that do not

43. pBR322
pBR322was one of the first versatile plasmid
vectors developed; it is the ancestor of many of the
common plasmid vectors used in biochemistry
laboratories.
pBR322contains an origin of replication(ori) and
a gene (rop) that helps regulate the number of
copies of plasmid DNA in the cell. There are two
marker genes: confers resistance to ampicillin,
and confers resistance to tetracycline. pBR322
contains a number of unique restriction sitesthat
are useful for constructing recombinant DNA.

44. pBR322
1.Origin of
replication
2.Selectable
marker
3.unique
restriction
sites

45. Enzymes
Restriction Enzymesand DNA LigasesAllow
Insertion of DNA Fragments into Cloning Vectors
1.Restriction endonuclease, RE
2.DNA ligase
3.Reverse transcriptase
4.DNA polymerase, DNA pol
5.Nuclease
6.Terminal transferase

46. Restriction enzymes cleave DNA
The same sequence of bases is
found on both DNA strands, but
in opposite orders. GAATTC
CTTAAG
This arrangement is called a
palindrome. Palindromesare
words or sentences that read the
same forward and backward.
form sticky ends: single
stranded ends that have a
tendency to join with each
other ( the key to
recombinant DNA)

47. Restriction Enzymes Cut DNA Chains at
Specific Locations
Restriction enzymesare endonucleases
produced by bacteria that typically
recognize specific 4 to 8bp sequences,
called restriction sites, and then cleave both
DNA strands at this site.
Restriction sites commonly are short
palindromic sequences; that is, the
restriction-site sequence is the same on
each DNA strand when read in the 5′ → 3′
direction.

48. Cut out the gene
Restriction enzymes

49. Restriction enzymes
Restriction enzymes are named after the
bacterium from which they are isolated
–For example, Eco RIis from Escherichia coli,
and Bam HIis from Bacillus amyloliquefaciens .
The first three letters in the restriction enzyme
name consist of the first letter of the genus (E)
and the first two letters of the species (co).These
may be followed by a strain designation (R)and
a roman numeral (I) to indicate the order of
discovery (eg, EcoRI, EcoRII).

50. Blunt ends or sticky ends
Each enzyme recognizes and cleaves a
specific double-stranded DNA sequence that
is 4–7 bp long. These DNA cuts result in
blunt ends(eg, HpaI) or overlapping (sticky)
ends(eg, BamH I) , depending on the
mechanism used by the enzyme.
Sticky ends are particularly useful in
constructing hybrid or chimeric DNA
molecules .

51. Results of restriction endonuclease digestion.
Digestion with a restriction endonuclease can result
in the formation of DNA fragments with sticky, or
cohesive ends (A) or blunt ends (B). This is an
important consideration in devising cloning
strategies.

53. Inserting DNA Fragments into Vectors
DNA fragments with either sticky endsor blunt
endscan be inserted into vector DNA with the
aid of DNA ligases.
For purposes of DNA cloning, purified DNA
ligaseis used to covalently join the ends of a
restriction fragment and vector DNA that have
complementary ends . The vector DNA and
restriction fragment are covalently ligated
together through the standard 3 → 5
phosphodiester bonds of DNA.
DNA ligase“pastes” the DNA fragments
together

54. Ligation of restriction fragments
with complementary sticky ends.

55. Identification of Host Cells
Containing Recombinant DNA
Once a cloning vector and insert DNA have
been joined in vitro, the recombinant DNA
molecule can be introduced into a host cell,
most often a bacterial cell such as E. coli.
In general, transformation is not a very
efficient way of getting DNA into a cell
because only a very small percentage of cells
take up recombinant DNA. Consequently,
those cells that have been successfully
transformed must be distinguished fromthe
vast majority of untransformed cells.

56. Identification of host cells containing
recombinant DNA requires genetic selectionor
screeningor 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.
Normally, a number of colonies of cells are first
selectedand then screenedfor colonies carrying
the desired insert.

57. Selection Strategies Use Marker Genes
(Primary screening)
Many selection strategies involve selectable
markergenes—genes whose presence can
easily be detected or demonstrated. amp
R
Selection or screening can also be achieved
using insertional inactivation.

58. A method of screening recombinants for inserted DNA fragments.
Using the plasmid pBR322, a piece of DNA is inserted into the unique
PstI site. This insertion disrupts the gene coding for a protein that
provides ampicillin resistance to the host bacterium. Hence, the
chimeric plasmid will no longer survive when plated on a substrate
medium that contains this antibiotic. The differential sensitivity to
tetracycline and ampicillin can therefore be used to distinguish clones
of plasmid that contain an insert.
insertional inactivation

59. Screening (Strategies)
1.Gel ElectrophoresisAllows Separation of
Vector DNA from Cloned Fragments
2.Cloned DNA Molecules Are Sequenced
Rapidly by the Dideoxy Chain-Termination
Method
3.The Polymerase Chain ReactionAmplifies a
Specific DNA Sequence from a Complex
Mixture
4.Blotting TechniquesPermit Detection of
Specific DNA Fragments and mRNAs with
DNA Probes

60. Southern blot technique can detect a specific DNA
fragment in a complex mixture of restriction fragments.
Radioactive isotope
Hybridization

61. Types of blotting techniques
Southern blotting
Southern blotting techniques is the first nucleic acid
blotting procedure developed in 1975 by Southern.
Southern blotting is the techniques for the specific
identification of DNA molecules.
Northern blotting
Northern blotting is the techniques for the specific
identification of RNA molecules.
Western blotting
Western blotting involves the identification of proteins.
Antigen + antibody

62. Expression of Proteins Using
Recombinant DNA Technology
Cloned or amplified DNA can be purified and
sequenced, used to produce RNA and protein, or
introduced into organisms with the goal of
changing their phenotype.
One of the reasons recombinant DNA technology
has had such a large impact on biochemistry is
that it has overcome many of the difficulties
inherent in purifying low-abundance proteins and
determining their amino acid sequences.

63. Recombinant DNA technology allows the
protein to be purified without further
characterization. Purification begins with
overproduction of the protein in a cell
containing an expression vector.
–Prokaryotic Expression Vectors
–Eukaryotic Expression Vectors

64. Prokaryotic Expression Vectors
Expression vectorsfor bacterial hosts are
generally plasmids that have been
engineered to contain appropriate
regulatory sequencesfor transcription and
translation such as strong promoters,
ribosome-binding sites, and transcription
terminators.

65. Eukaryotic proteins can be made in bacteria by
inserting a cDNA fragmentinto an expression
vector . Large amounts of a desired protein can be
purified from the transformed cells.
In some cases, the proteins can be used to treat
patients with genetic disorders.
For example, human growth hormone, insulin, and
several blood coagulation factorshave been produced
using recombinant DNA technology and expression
vectors.

66. Expression of Proteins in Eukaryotes
Prokaryotic cells may be unable toproduce
functional proteins from eukaryotic genes
even when all the signals necessary for gene
expression are present because many
eukaryotic proteins must be post-
translationally modified.

67. Several expression vectorsthat function in
eukaryotes have been developed.
These vectors contain eukaryotic origins of
replication, marker genesfor selection in
eukaryotes, transcription and translation
control regions, and additional features
required for efficient translation of
eukaryotic mRNA, such as polyadenylation
signals and capping sites.

69. Recombinant DNA Technology
1.The basic concepts for recombinant
DNA technology
2.The basic procedures of recombinant
DNA technology
3.Application of recombinant DNA
technology

70. Applications of Recombinant
DNA Technology

71. 1.Analysis of Gene Structure and
Expression
2.Pharmaceutical Products
–Drugs
–Vaccines
3.Genetically modified organisms (GMO)
–Transgenic plants
–Transgenic animal
4.Application in medicine