GENE LIBRARY

By,Pillai Aswathy viswanathPG 2 BotanySt. Thomas college kozhencherry

INTRODUCTION

In molecular biology, a gene library is a collection of DNA that is stored and propagated in a population of micro-organisms .

so that there is a high probility of finding any particular piece of the DNA in the collection

There Are Mainly 2 Types Of gene Libraries 

Gene Library

cDNA Library Genomic library

cDNA library :- The cDNA library contain

only complementary DNA molecules synthesized from mRNA molecules in a cell

It represents only the expressed part of the genome and contain only coding sequences

cDNA has only coding sequences therefore can be directly expressed

in prokaryotic system.

Genomic library :- The genomic library

contains DNA fragment representing entire genome of an organism ,having both coding and non coding regions.So the genes taken from genomic library is difficult in prokaryotic system like bacteria due to absence of splicing mechanism.

Procedure for the construction of a genomic library using phage λ system

1. Isolation Of DNA To create a genomic library, a

researcher begins by  isolation of DNA from the organisms.

Lysis of cells with detergent containing lysis buffer.

Incubation of cells with digestion buffer containing protease-K, SDS to release genomic DNA from DNA-protein complex.

2.Purification Of DNA Purification of genomic DNA

with phenol: chloroform mixture. Chloroform: phenol mixture

Transfer the nucleic acid sample to a polypropylene tube and add an equal volume of phenol: chloroform.

It has two phases, aqueous phase and organic phase.

In this step, phenol denatures the remaining proteins and keep the protein in the organic phase.

Genomic DNA present in aqueous phase is again precipitated with absolute alcohol.

3.Generation Of Genomic DNA Into Suitable Small Size Fragments.

The purified DNA consist of extremely long strands and needs to be cut into desirable fragment sizes.

The DNA therefore is digested with restriction enzymes which cut the DNA at specific sequences

The restriction enzyme cut the DNA into 1000s of smaller fragments , each of which contain one or more gene

Each fragment is different and have unique DNA sequence

If a organism has a genome size of 2x107 kb and an average size of the fragment is 20kb,

4.Cloning Into The Suitable Vector Cloning vector are those DNA

molecule that carry a foreign DNA fragments when inserted into it

The suitable vector to prepare the genomic library can be selected based on size of the fragment of genomic DNA

For constructing genomic DNA library, we mostly preferred λ phage vectors

λ phage as a higher transformation efficiency about 1000 times higher compared to a plasmid.

λ phage has high cloning and packaging efficiency and is easy to handle and store as compared to plasmid vectors

A 50kb can be inserted into the λ phage The vector as to maintain its lytic

growth.

λ-phage can be digested with the same restriction enzyme used for digestion of DNA

The vector arms and genomic fragments are annealed together

T4 DNA ligase is used to ligate the selected DNA sequence

5.TRANSFORMATION IN SUITABLE HOST Once the recombinant

DNA molecule has been constructed, it has to be introduced in to suitable host amplification. This is called gene cloning

Phage λ contains a proteinaceous head and a long tail attached to the head

In the head it possesses 50 genes in its 50 kb

On attachment with tail to cell wall of E.coli it injects its linear DNA in to the cell

clones are transformed in a suitable host to get colonies

When the bacteria have taken up the DNA,the entire collection of cells and DNA represents a human genome library

REFERENCE Dubey R.C,A textbook of biotechnology,

(2004),published by S.Chand and company LTD.

Chawla H.S,Introduction to plant biotechnology,(2000).oxford and IBH publishing Co.Pvt.Ltd.New Delhi

Rastogi S.C,shivani Rastogi,introduction to biotechnology , CBS publishers and distributors

http://www.biotechnologyforums.com/thread1830.html

http://www.majordifferences.com/2013/11/differencebetweengenomicandcdna