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Gene Libraries
It is collection of clones that represents the completegenome of
an organisms
For construction of gene or any another type of
library mainly four things are required1 .Resource of
DNA/gene
2. Vector DNA
3.Enzymes
4. Suitable host
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Types of Gene libraries
A DNA library is a comprehensive collection ofcloned DNA
fragments which includes one with thegene of interest
There are two types of libraries: Genomic libraries
cDNA libraries
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cDNA LIBRARIES
In order to synthesize double-stranded DNA, RNase H
mustfirst
be used RNase H is derived fromE. coli and recognizes RNA-DNA
hybrids
It digests the RNA into small fragments E. coli DNA polymerase I
uses the original cDNA template to synthesize the complementary
strand,replacing the RNA except for a small piece at the 5 end The
DNA is made continuous by ligating with DNA ligase
Its also possible to attach artificial sites to the cDNA
ends
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CONSTRUCTION of cDNA LIBRARY
A cDNA library is a collection of all the expressed DNA of a
particular celltype or tissue.
Eg., A cDNA library from the bone marrow cells will have many
clones withthe cDNA for
hemoglobin alpha and beta chains. The cDNA libraries are
advantageous since they don't have the intron
sequences which is present in the genomic libraries cDNA clones
find application where bacterial expression of the foreign
DNA is necessary, either as a prerequisite for detecting the
clone or because the
polypeptide product is the primary objective cDNA cloning can be
used to study the temporally regulated gene
expression in development, or tissue specific
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CONSTRUCTION of cDNA LIBRARY
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CONSTRUCTION of cDNA LIBRARY(continued)
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CONSTRUCTION of cDNA LIBRARY(continued)
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(continued)
Double stranded cDNA copies of mRNA withEco R1 cohesive ends are
ready to ligate intoa bacteriophage lambda vector cut withEco
R1
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GENOMIC LIBRARIES
Creating a genomic library consists of digesting theentire
genome of a cell with a restriction enzyme inorder to produce a
large number of DNA fragments
Some fragments will contain genes, others willcontain portions
of genes and still others will containnoncoding DNA.
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GENOMIC LIBRARY CONSTRUCTION
Vector DNA (bacteriophage lambda)
Lambda has a linear double stranded DNA genome.
The left and the right arms are essential for the
phage replication cycle The internal fragment is dispensable
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Advantage of genomic library
The regulatory sequences flanking the coding portionof the
gene
The non coding intervening sequences
The various members of a multigenes family whichoften lies close
together in the genome
The evolution of DNA sequences, includingduplication and
rearrangement as seen in
comparasion of the DNA of different species
The interspersion of transposable elements
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Disadvantage of genomic library
The Eukaryotes have intron sequence
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