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Thursday, August 19, 2010

PowerPoint Presentation On Genomic Library

PPT On Genomic Library


Genomic Library Presentation Transcript:
1. Genome - An Introduction
The total DNA present in the nucleus of each cell of an organism is its Genome. It comes from the terms Gene and Chromosome. It corresponds to all the organism’s bases: A,T,C,G. Our genome is a chain of 3.4 billion ‘pearls’. The genomes of two different people differ on an average by on ‘pearl’ in one thousand. Genes represent only about 5% of the total human genome. The role of all the non-coding sequences that make up the remaining 95% is today unknown, but researches have been able to assign functions to some of those sequences: some regulate DNA replication and transcription; others contribute to the chromosomal structuring. The Human Genome Project, launched in the early 1990s has succeeded in the completing the draft of DNA sequence(2003) of our entire genome. Genome is divided into chromosomes, chromosomes contain genes, and genes are made of DNA.

2. The word “genome” was coined in about 1930, however at that time the scientists had very little information about the real meaning of the word “Genome”. Each one of the earth’s species has its own distinctive genome. Genomes belong to species,& they also belong to individuals. Every Giraffe on the African Savanna has a unique genome, as does every elephant, acacia tree, & ostrich. Unless individuals are identical twins, their genomes are different from each other & every other person on earth-in fact it differs for every single individual who has ever lived. The entire world is full of Genomes. A genome is the information that affects every aspect of our behavior & physiology. Cooking dinner, digesting food talking, sleeping, reading – the genome plays a role in all these things. Thus studying the genome gives us insights into why some people live longer than others, why some die of heart disease and others of cancer, why some people have trouble keeping weight on while others have trouble keeping it off and so on.

3. Gene Library
A gene library is a collection of different DNA sequences from an organism each of which has been cloned into a vector for ease of purification, storage and analysis. There are essentially two types of gene library that can be made depending upon the source of the DNA used. If the DNA is genomic DNA, the library is called a genomic library. If the DNA is a copy of an mRNA population, that is cDNA, then the library is called a cDNA library. Size of the gene library A gene library must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence. This value can be calculated if the genome size and the average size of the insert in the vector are known. Construction of libraries For making libraries, genomic DNA, usually prepared by protease digestion and phase extraction, is fragmented randomly by physical shearing or restriction enzyme digestion to give a size range appropriate for the selected vector. Often combinations of restriction enzymes are used to partially digest the DNA. Vectors Plasmids, Lambda phage, cosmid, BAC or YAC( yeast artificial chromosome) vectors can be used to construct genomic libraries, the choice depending on the genome size. The upper size limit of these vectors is about 10, 23, 45, 350, & 100 kb respectively. The genomic DNA fragments are ligated to the prepared vector molecules using T4 DNA ligase.

4. Genomic DNA Libraries
These libraries are made from genomic DNA (all the DNA found in the organism’s nuclei). Genomic DNA molecules are very large (each chromosome in the nucleus is one such DNA molecule), so they must be fragmented into small pieces to insert into vectors. This is done through digestion using one or more appropriate restriction endonucleases, mechanical shearing, or a combination of the two processes. The DNA is then ligated into the vector, which could be a plasmid, a cosmid (more often) or a viral chromosome. cDNA Libraries These libraries are made from cDNA (complimentary DNA), which are DNA copies of mRNA molecules. To make cDNA, mRNA is isolated from a tissue or whole organism, and DNA is copied from the mRNA template using an enzyme called reverse transcriptase. This enzyme works like a DNA polymerase, except that it uses RNA as a template instead of DNA. The resulting cDNA molecules are then engineered so that they have restriction enzyme recognition sites at each end of every molecule, which allows them to be digested and inserted into a vector.

5. Genomic Libraries
Definitions A library produced when the complete genome of a particular organism is cleaved into thousands of fragments, and all the fragments are cloned by insertion into a cloning vector. A form of gene library containing the complete DNA sequences present in the genome of a given organism. A collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. A set of thousands of DNA segments from a genome, each carried by a plasmid or a phage.

6. Construction of Genomic Libraries
The purpose of genomic library construction is to have an organism’s genome cloned as small fragments into separate vectors. Ideally the entire genome is represented: i.e., to say, the sum of the different fragments equals the entire genome. In this way specific groups of genes can be analyzed and isolated. The construction of a genomic library begins with cleaving the genome into small pieces by a restriction endonuclease. These genomic fragments are then either cloned into vectors & introduced into a microbe or packed into phage particles that are used to infect the host. In either case, many thousands of different clones- each with a different genomic DNA insert –are created. Therefore each clone will act as a “book” in this “library” of DNA fragments. If the genomic library has been inserted into a microbe that expresses the foreign gene, it may be possible to assay each clone for a specific protein or phenotype.

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