Gene Libraries

It is collection of clones that represents the complete

genome of an organisms For construction of gene or any another type of library mainly four things are required 1 .Resource of DNA/gene 2. Vector DNA 3.Enzymes 4. Suitable host

Types of Gene libraries

A DNA library is a comprehensive collection of

cloned DNA fragments which includes one with the gene of interest There are two types of libraries: Genomic libraries cDNA libraries

cDNA LIBRARIES

first be used RNase H is derived from E. 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

In order to synthesize double-stranded DNA, RNase H must

CONSTRUCTION of cDNA LIBRARY

A cDNA library is a collection of all the expressed DNA of a particular cell

type or tissue. Eg., A cDNA library from the bone marrow cells will have many clones with the 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

CONSTRUCTION of cDNA LIBRARY

CONSTRUCTION of cDNA LIBRARY (continued)

CONSTRUCTION of cDNA LIBRARY (continued)

(continued)
Double stranded cDNA copies of mRNA with

Eco R1 cohesive ends are ready to ligate into a bacteriophage lambda vector cut with Eco R1

GENOMIC LIBRARIES
Creating a genomic library consists of digesting the

entire genome of a cell with a restriction enzyme in order to produce a large number of DNA fragments Some fragments will contain genes, others will contain portions of genes and still others will contain noncoding DNA.

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

Advantage of genomic library

The regulatory sequences flanking the coding portion

of the gene The non coding intervening sequences The various members of a multigenes family which often lies close together in the genome The evolution of DNA sequences, including duplication and rearrangement as seen in comparasion of the DNA of different species The interspersion of transposable elements

Disadvantage of genomic library

The Eukaryotes have intron sequence

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