Transcript PPT1 - Ycmou

Online Counselling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
OC-SBT093-U01-01
Introduction
Programmes and Courses
 SEP – SBT093 – CP1-UN 01
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School of Science and Technology, Online Counselling Resource…
Credits
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Academic Inputs by
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Arun Punaji More.
M.Sc. (Microbiology)
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Experience: 11 Years.
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[email protected]
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School of Science and Technology, Online Counselling Resource…
How to Use This Resource
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Counsellor at each study centre should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counselling.
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Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.
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Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.
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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.
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Appear several times, for all the Self-Tests, available for this course.
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Student can use handouts for last minutes preparation just before end
exam.
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School of Science and Technology, Online Counselling Resource…
Learning Objectives
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After studying this module, you should be able to:
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Define the term “Genomic library”
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Describe the various methods of preparation of genomic library
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Describe uses of genomic library in genetic engineering
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School of Science and Technology, Online Counselling Resource…
Introduction-1
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Isolation of gene of interest is based upon
the successful cloning that gene and its
subsequent identification with the help of
some selectable markers such as auxotrophic
mutations or antibiotic resistance.
Many times it is practically difficult to isolate
a single gene of our interest due to absence
of suitable selectable markers as mentioned
above.
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School of Science and Technology, Online Counselling Resource…
Introduction-2
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In such case all the genes of a genome
is cloned and then gene of interest is
identified by a suitable radio-labeled
DNA probe.
Thus a set of clones of all the genes
from a genome can be prepared
constituting the genome library.
In this module students shall learn about
the different methods of preparation of
genome library and isolation and
identification of desired gene or genes
from this library.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-1
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Genomic library is defined as a set of clones
of all the genes of a genome of a organism.
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The size of genomic library is determined by
the size of DNA fragment that can be
inserted into cloning vector.
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The size of genomic library or the number of
clones in a genomic library can be
determined by following formula:
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N = ln(1-P)/ln(1-a/b)
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School of Science and Technology, Online Counselling Resource…
Genomic Library-2
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In this formula i.e. N = ln(1-P)/ln(1-a/b)
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N = number of clone in a genomic library
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a = average size of DNA fragment.
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b = total size of genome.
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p = probability of presence of any gene.
© 2007, YCMOU. All Rights Reserved.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-3
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Preparation of genomic library involve following
steps:
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Purification of total cell DNA
Partial digestion by restriction enzymes to
produce DNA fragments.
Cloning or insertions of these DNA fragments
into suitable vectors such as lambda
replacement vector, a cosmid, bacterial
artificial chromosome or yeast artificial
chromosome.
Isolation of recombinant clones carrying
separated gene of the genome; this is
genomic library.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-4
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Purification of total cell DNA:
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Purification of total DNA is the first step in
preparation of genomic library.
To obtain total cell DNA in pure form one has
to obtain
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Pure cell culture and harvestation cell mass
Preparation of cell free extract
Removing the other cell ingredients to obtain
DNA in pure form
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School of Science and Technology, Online Counselling Resource…
Genomic Library-5
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Purification of total cell DNA:
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After growing and harvesting the pure culture;
the cell mass is subjected to various
techniques to obtain the cell free extract
which contain all the cell content from which
DNA is purified.
First cell free extract is treated with protease
enzymes such as pronase and proteinase K to break
down proteins into smaller polypeptide units so that
they can be easily precipitated out by phenol and
chloroform treatment.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-6
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Purification of total cell DNA:
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Next this enzyme treated cell free extract is
deproteinized by mixing with phenol solution or
phenol and chloroform mixture in 1:1 ratio; the
proteins get precipitated out leaving the DNA and
RNA in the aqueous solution.
Now to obtain the DNA free from RNA in the
aqueous solution, RNA is degraded by enzyme
ribonuclease into ribonucleotide subunits.
DNA is purified further by ultracentrifugation and
concentrated by the ethanol precipitation at -20˚C.
DNA can also be purified by ion-exchange
chromatography.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-7
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Preparation of DNA fragments for
cloning:
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The purified genomic DNA is then partially
digested with restriction endonucleases.
For partial digestion of genomic DNA, only
those restriction endonucleases are used
which cut DNA molecules in very precise and
reproducible manner.
Some of the widely used restriction
endonucleases are
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EcoRI
BamHI
HindIII
TaqI
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School of Science and Technology, Online Counselling Resource…
Genomic Library-8
 Preparation of genome
Figure shows steps involved
in Genomic library.
library
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School of Science and Technology, Online Counselling Resource…
Genomic Library-9
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Insertion of DNA fragments into vectors:
Once number of fragments are obtained by
restriction endonucleases digestion, these
fragments are then inserted into suitable
vectors such as:
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λ replacement vector
Cosmid
Bacterial artificial chromosome (BAC) or P1
vector
Yeast artificial chromosome (YAC)
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School of Science and Technology, Online Counselling Resource…
Genomic Library-10
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Isolation of recombinant clones:
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The chimeric vectors with inserted genes are
allowed to infect their suitable host bacteria
which are grown on a suitable medium.
A isolated pure colony of bacteria represent a
clone of a isolated gene from the genome.
All the colonies thus obtained represent the
set of all genes from the genome and
therefore a library of the genome.
The library of these genes are then searched
for a desired gene or genes by radio labeled
DNA probe using DNA–DNA hybridization
technique.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-11
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cDNA Library:Preparation of cDNA
Library using mRNA
cDNA library make isolation
of a desired gene easy
and less tedious involving
less steps than involved
in genome library.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-12
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Probing of genomic library:–
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Plaque and colony hybridization probing
Abundancy probing on cDNA library
Heterologous probing
Probing with oligonucleotide probes
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School of Science and Technology, Online Counselling Resource…
Genomic Library-13
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Plaque and colony hybridization
probing:–
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Recombinant DNA is detected directly in
bacterial colonies and bacteriophage
plaques.
The colonies or plaques are transfered
onto nitrocellulose papers and treated to
denature the DNA molecules and allowed
to react with radio-labeled probes.
The membrane is then autoradiographed
and position of colony or plaque
containing the desired recombinant DNA
is fixed and isolated in pure form for
further study.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-14
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Abundancy probing on cDNA library:–
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Individual clones from the cDNA library
itself is used as probe to screen the
library .
The clone which hybridizes with
maximum clones in the library is the
desired clone present in the cDNA library.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-15
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Heterologous probing:–
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Uses known gene from one species to
identify the same gene in another related
species by the DNA hybridization
technique.
The heterologous probing uses the
principle that there is nucleotide
similarity between two genes encoding
for the same protein in two different
organisms due to conservation of gene
structure during evolution.
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School of Science and Technology, Online Counselling Resource…
Genomic Library-16
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Probing with oligonucleotide probe:–
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The nucleotide sequence of a gene whose
protein has been sequenced can be
deciphered and used to construct a DNA
fragment chemically in laboratory.
This chemically synthesized DNA
fragment of known nucleotide sequence
is called as oligonucleotide probe.
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School of Science and Technology, Online Counselling Resource…
What We Learn…..
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Definition of genomic library
Methods of preparation of genomic
library.
Principle of preparation of genomic
library.
Technique of identification of the clone
containing desired gene.
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School of Science and Technology, Online Counselling Resource…
Critical Thinking Question
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Why genomic library is constructed
usually for less complex genome of
microorganisms?
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School of Science and Technology, Online Counselling Resource…
Tips for Critical Thinking Question
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Manageable size of simple genome
Produce less clones that can be analyze
easily.
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School of Science and Technology, Online Counselling Resource…
Study Tips
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Book:
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Title: Molecular Biology Of The Gene
Author: J. D. Watson, Becker, Bell, etc
Publication: Pearson Education
Book:
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Title: Gene Cloning and DNA analysis
– An Introduction
Author: T. A. Brown
Publication: Blackwell Publishing
© 2007, YCMOU. All Rights Reserved.
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End of the Presentation
Thank You !
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