Transcript PPT1 - Yashwantrao Chavan Maharashtra Open University

Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
OC-SBT053-CP1-01
Introduction
Programmes and Courses
 SEP – SBT053 – Unit 01
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Credits
 Academic Inputs by
 Mrs. Rasika Bhore
 M.sc (Microbiology)
 [email protected]
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How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
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School of Science and Technology, Online Counseling Resource…
Learning Objectives

After studying this module, you should be able to:

Discuss cloning or recombinant DNA technology.

Describe the importance of restriction enzyme in cloning &
mechanism performed by restriction enzymes.
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School of Science and Technology, Online Counseling Resource…
Introduction
 Genetic engineering is a laboratory technique used to change
the DNA of living organisms.
 Genetic engineering succeded in:-
 Improving crop technology.
 Manufacture of erythropoietin in ovary cells, and
 Production of new types of experimental mice such as
the oncomouse.
 One of the best known applications of genetic engineering is
the creation of genetically modified organisms (GMOs) such as
foods and vegetables that resist pest and bacterial infection
and have longer freshness than otherwise.
 The biotechnological applications of GM, for example oral
vaccines produced naturally in fruit, at very low cost.
 The important tools of genetic engineering are Enzymes.
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Cloning
 ‘Clone’ is an identical copy.
 Cloning is isolation of single types of cells & allowed it to
reproduce & create a population of identical cells.
 "DNA cloning” involves transfer of a DNA fragment of
interest from one organism to a self-replicating genetic
element such as a bacterial plasmid (vector).
 The fragment of chromosomal DNA is joined with its
cloning vector is called a "recombinant DNA molecule."
 Further this modified DNA is replicated millions of times to
increase in cell number & for the creation of multiple copies
of the cloned DNA in each cell.
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Steps of Cloning
1.
2.
3.
4.
5.
Cutting DNA at precise
locations. This is done with
the help of restriction
enzymes.
Selection of small molecules
of self-replicating DNA i.e.
plasmids.
Joining two fragments
covalently.
Moving recombinant DNA
from the test tube to a host
cell.
Selection of host cells
containing recombinant DNA.
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Restriction Enzyme
 A restriction enzyme or restriction endonuclease
is an enzyme that cuts double-stranded DNA.
 The enzyme makes two incisions, one through each
of the sugar-phosphate backbones of the double
helix without damaging the nitrogenous bases.
 The term restriction comes from the fact that these
enzymes were discovered in bacterial strains that
appeared to be restricting the infection by certain
bacteriophages.
 Restriction enzymes therefore are believed to be a
mechanism evolved by bacteria to resist viral
attack and to help in the removal of viral
sequences.
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Discovery And Application
 The 1978 Nobel Prize in Medicine was
awarded to Daniel Nathans, Werner
Arber and Hamilton Smith for the
discovery of restriction endonucleases,
leading to the development of
recombinant DNA technology.
 The first practical use of their work was
the manipulation of E. coli bacteria to
produce human insulin for diabetics.
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Types of Restriction Enzyme
 There are three groups of restriction enzyme that
vary in the way recognise their restriction sites and
where they cut the DNA:
 Type I restriction enzymes cut DNA at random
sites that can more than 1000 nucleotides after
the recognition site and requires ATP.
 Type II restriction enzymes cut DNA at the
recognition site and for this reason are most
often used in scientific experimentation & don’t
require ATP.
 Type III restriction enzymes cut DNA about 2030 base pairs after the recognition site and
requires ATP.
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Some Restriction Enzymes……..
Enzyme
Organism from which
derived
Target sequence
(cut at *)
5' -->3'
AvaI
Anabaena variabilis
C* C/T C G A/G G
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
GG*CC
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Kpn I
Klebsiella pneumoniae
GGTAC*C
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
Xma I
Xanthamonas
malvacearum
C*CCGGG
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Action Of Restriction Enzyme
 Restriction enzymes recognize a specific
sequence of nucleotides and produce a double
stranded cut in the DNA that prevents the
phage from replicating.
 While recognition sequences vary widely, with
lengths between 4 and 8 nucleotides, many of
them are palindromic; that is, the sequence on
one strand reads the same in the same
direction on the complementary strand.
5'- G A A T T C -3‘
3'- C T T A A G -5'
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Sticky Ends
 Some restriction endonucleases make staggered cuts on the
two DNA strands, leaving 2-4 nucleotides of one strand
unpaired at each resulting end.
 These unpaired strands are referred as sticky ends, as they
can form pair with each other or with complementary sticky
ends of other DNA fragments.
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Eco RI Produces sticky Ends……..
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Blunt Ends
Some restriction endonucleases cleave both strands
of DNA at the opposing phosphodiester bonds,
leaving no unpaired bases on the ends, often called
blunt ends.
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Sma I Produces Blunt Ends………..
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Exonuclease
 Exonucleases are enzymes found as individual enzymes, or
as parts of larger enzyme complexes that cleave nucleotides
one at a time from an end of a polynucleotide chain.
 These enzymes hydrolyze phosphodiester bonds from either
the 3' or 5' terminus of a polynucleotide molecule.
 The difference between exonuclease & endonuclease is exonuclease progressively splits off single nucleotides from
one end of DNA or RNA & endonuclease splits DNA or RNA at
internal sites.
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What We Learn………….
 To "clone a gene," a DNA fragment containing
the gene of interest is isolated from
chromosomal DNA using restriction enzymes
and then united with a plasmid that has been
cut with the same restriction enzymes.
 Restriction enzymes have been found in
bacterial strains.
 Type 2 restriction enzymes cut DNA at the
recognition site and for this reason are most
often used in scientific experimentation.
 Restriction enzymes create either sticky or
blunt ends in strand.
 Exonuclease cleave one nucleotide at one time.
School of Science and Technology, Online Counseling Resource…
Critical Thinking Questions
 Restriction enzymes present in bacterial species
recognize particular nucleotide sequence & cleave
the viral DNA at that site. If the bacterial DNA
posses the same sequence, will it cleaved by
restriction enzyme? Why?
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School of Science and Technology, Online Counseling Resource…
Tips For Critical Thinking Questions
 DNA methylation.
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School of Science and Technology, Online Counseling Resource…
Study Tips
 Book

Title: Principles of
Biochemistry

Author: David L. Nelson
 Book

Title: Genes VIII

Author: Benjamin Lewin
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School of Science and Technology, Online Counseling Resource…
Study Tips
www.en.wikipedia.org
Genetic Engineering
www.vivo.colostate.edu
Biology & Activity of Restriction Endonuclease
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End of the Presentation
Thank You