Genomic library : Genomic library Presented by Pratiksha srivastava B.tech-M.tech (BT) Genome – An Introduction : Genome – An Introduction Genome : “The Book Of Life” The word “genome” was coined in about 1930. The total DNA present in the nucleus of each cell of an organism is its Genome . It comes from the terms Gen e and Chromos ome . It corresponds to all the organism’s bases: A,T,C,G. Genes represent only about 5% of the total human genome, and remaining 95% is unknown i.e. non coding sequences, but some have been assign functions : some regulate DNA replication and transcription; others contribute to the chromosomal structuring. Genome is divided into chromosomes, chromosomes contain genes, and genes are made of DNA. Gene Library : Gene Library A gene library is a collection of different DNA sequences from an organism,which has been cloned into a vector for ease of purification, storage and analysis. There are two types of gene library that can be made depending upon the source of the DNA used. genomic library. cDNA library. Types of GENE library: Types of GENE library The genomic library contains DNA fragments representing the entire genome of an organism. The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. Genomic Library : Genomic Library Are made from total nuclear DNA of an organism or species. DNA is cut into clonable size pieces as randomly possible using restriction endonuclease Genomic libraries contain whole genomic fragments including gene exons and introns, gene promoters, intragenic DNA,origins of replication, etc PowerPoint Presentation: Construction of Genomic Libraries There are following main steps in gene cloning: 1. Isolation of genomic DNA and vector. 2. Cleavage of Genomic DNA and vector by Restriction Endonucleases. 3. Ligation of fragmented DNA with the vector. 4. Transformation of r-DNA in the bacterial cell. 5. Amplification of the r-DNA in bacterial cells. PowerPoint Presentation: Step1:- Extraction of genomic DNA PowerPoint Presentation: Step2:- Cut with a restriction endonuclease enzyme PowerPoint Presentation: Step3:-Ligate the gene with the vector which cleaves by same enzyme PowerPoint Presentation: Step4:-Transfer (transform) into bacteria Cells which are able to undergo this treatment are termed as competent cells . CaCl 2 causes DNA to precipitate on the outside walls of bacterial cells. PowerPoint Presentation: Step5:- Amplification E.coli cells are grown in an agar medium containing ampicillin or tetracyclin at 37°C. CONSTRUCTION OF GENOMIC LIBRARY: CONSTRUCTION OF GENOMIC LIBRARY Screening of genomic library: Screening of genomic library Once the genomic library has been created, it is screened to identify the genes of interest. One of the most common library screening technique is called colony hybridization. In the process of library construction, phage vectors are used then the process of identification of genes of interest involved is the plaques hybridization . colony hybridization: colony hybridization Colony Hybridization is the screening of a library with a labeled probe (radioactive, etc.) to identify a specific sequence of DNA, RNA, enzyme, protein, or antibody. Plaque hybridization : Plaque hybridization The plaques are screened by a technique based on the hybridization of oligonucleotide probe to target DNA. In this case, DNA is transferred directly from the Petri dish to the filter, which is then incubated with labeled probes. Applications of Genomic Library -: Applications of Genomic Library - 1. Genomic library construction is the first step in any DNA sequencing projects. 2. Genomic library helps in identification of the novel pharmaceutically important genes. 3. Genomic library helps in identification of new genes which were silent in the host. 4. It helps us in understanding the complexity of genomes. PowerPoint Presentation: 5. Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering . 6. Study of the function of regulatory sequences in vitro. 8. Study of genetic mutations in cancer tissues. 9.Create cDNA libraries to determine what genes are being expressed at a particular time. PowerPoint Presentation: Thank you PowerPoint Presentation: Queries ?