Recombinant Dna technology, Restriction Endonucleas and Vector

RDNA Technology

1. Recombinant DNA
Technology
By
Dr. Priti D.Diwan
Assistant Professor
Department of Zoology
J.D.Patil Sangludkar Mahavidyalay Daryapur.

2. Recombinant DNA and
Gene Cloning
RecombinantDNA(rDNA)isaformofartificial
DNAthatiscreatedbycombiningtwoormore
DNAsequencesoriginatingfromdifferent
organism.
RecombinantDNAtechnologyisatechnology
whichallowsDNAtobeproducedviaartificial
means.Theprocedurehasbeenusedtochange
DNAinlivingorganismsandmayhaveevenmore
practicalusesinthefuture.

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

4. Stanley N. Cohen (1935–) (top)
andHerbert Boyer (1936–)
(bottom), who constructed the
first recombinant DNA using
bacterial DNA and plasmids.
Stanley N. Cohen , who
received the Nobel Prize in
Medicine in 1986 for his
work on discoveries of
growth factors.

5. 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.

6. 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.

7. Brief Introduction

8. The basic concepts for
recombinant DNA technology

9. 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!

10. 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.

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

12. The basic procedures of
recombinant DNA technology

13. 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.

14. 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.

15. 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

16. 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.

17. 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.

18. 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.

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

20. Applications of Recombinant
DNA Technology

21. 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

23. •Insulin
–Hormone required to
properly process sugars
and fats
–Treat diabetes
–Now easily produced by
bacteria
•Growth hormone
deficiency
–Faulty pituitary and
regulation
–Now easily produced by
bacteria

24. 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

25. 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

27. 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.

28. Transgenic animals
Green fluorescence Red fluorescence

29. Transgenic animals

30. A transgenic
mouse
Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat growth hormone

31. Farm Animals and “Pharm”
Animals
These transgenic sheep
carry a gene for a
human blood protein
–This protein may help in
the treatment of cystic
fibrosis

32. Other benefits of GMOs
Disease resistance
There are many viruses, fungi, bacteria that cause plant
diseases
“Super-shrimp”
Cold tolerance
Antifreeze gene from cold water fish introduced to
tobacco and potato plants
Drought tolerance & Salinity tolerance
As populations expand, potential to grow crops in
otherwise inhospitable environments

33. What are restriction enzymes?
•Molecularscissorsthatcutdouble
strandedDNAmoleculesatspecific
sequencethatsequenceiscalledas
recognitionsequenceorsite.
•Foundnaturallyinawidevarietyof
prokaryotes
•AnimportanttoolformanipulatingDNA.
•3,000enzymeshavebeenidentified,
around200haveuniqueproperties,many
arepurifiedandavailablecommercially

34. Discovery
•Arbor and Dussoix in 1962 discovered that
certain bacteria contain Endonucleases
which have the ability to cleave DNA.
•In 1970 Smith and colleagues purified and
characterized the cleavage site of a
Restriction Enzyme.
•Werner Arbor, Hamilton Smith and Daniel
Nathans shared the 1978 Nobel prize for
Medicine and Physiology for their
discovery of Restriction Enzymes.

35. Types of Restriction Endonucleases
TypeI-Mostcomplexandbifunctionali.edorestriction
aswellasmodificationactivity.CleaveDNAatrandom
sitehencenotusedinrDNAtechnology.EgEcoK,EcoB
TypeII-simple,cleaveDNAatspecificrecognition
sequencesite(Palindromic).Therearemorethan350
TypeiiREwith100differentrecognitionsitesareuptill
known.TheyrequiresMg2+Ionsforcleavage.Only
TypeIIREareusedinrDNAtechnology.EcoR1,HindIII
TypeIII-TheseareintermediatebetweenTypeI&II
RE.Recognitionsiteisasymmetricalsequenceof5-7
BP.Eg.EcoP1,EcoP15,HintF3

36. Restriction Endonucleasesnomenclature
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Mechanism of Action
Restriction Endonuclease scan the length of the DNA , binds to the DNA
molecule when it recognizes a specific sequence and makes one cut in each
of the sugar phosphate backbones of the double helix –by hydrolyzing the
phoshphodiester bond. Specifically,the bond between the 3’ O atom and the P
atom is broken.

37. Recognition sites have symmetry (palindromic)
Able was I
I saw elbA

38. Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion -“sticky ends”
Some enzymes cut in a direct fashion –“blunt ends”

43. Inmolecularcloning,avectorisaDNAmoleculeusedasa
vehicletoartificiallycarryforeigngeneticmaterialintoanother
cell,whereitcanbereplicatedand/orexpressed.
VECTOR

45. CLONING VECTORS
•CloningvectorsareDNAmoleculesthatareusedto"transport"cloned
sequencesbetweenbiologicalhostsandthetesttube.
•Mostvectorsaregeneticallyengineered.
•AvectorisusedtoamplifyasinglemoleculeofDNAintomanycopes.
Cloningvectorssharecommonproperties:
1.Abilitytoreplicate.
2.Easytoisolateandpurify
3.Containageneticmarkerforselection.
4.UniquerestrictionsitestofacilitatecloningofinsertDNA.
5.MinimumamountofnonessentialDNAtooptimizecloning