Gene Libraries

Gene Libraries- Genomic library, construction of genomic library, screening. applications. cDNA library, construction of cDNA library, screening, applications.

by MSC MICROBIOLOGY STUDENT- Falguni Agrawal, Barkatullah University, Bhopal

1. GENE LIBRARIES ~ By Falguni Agrawal

2. GENES (1865) Gregor Mendel – Each heritable property of an organism is controlled by a “Factor”(later called as “gene”) (1903) Walter Sutton – Proposed that these factors reside on chromosomes. (1909) Wilhelm Johannsen – Coined the term “Gene” to describe the functional unit of heredity. (1910) T. H. Morgan – Developed techniques for gene mapping (1944) Avery, Macleod, McCarty and (1952) Hershey & chase – proved DNA as a genetic material Each set of chromosomes contain 50,000 to 100,000 genes carried in 3 billion nucleotide pairs of DNA.

3. GENE LIBRARY Based on from where the segments of DNA are derived, gene library can be of 2 types: Genomic library cDNA library The first ever Gene library was constructed by Fredrick Sanger in 1977 of the bacteriophage , phi X 174 . GENE LIBRARY Collection of bacterial cells Each bacterial cell Vector Single segment of DNA Genomic DNA cDNA

4. GENOMIC LIBRARY Genome - an organism’s complete set of DNA, including all of its genes. Genomic library - collection of cloned, restriction enzyme digested DNA fragments containing at least one copy of every DNA sequence present in genome of an organism. Factors to be considered and requirements needed before constructing a genomic library : a) Organism of interest b) Restriction Enzymes c) Appropriate vector needs to be chosen based on the organism’s genome size. d) Ligase enzyme e) The necessary number of recombinant DNA molecules has to be calculated.

5. CONSTRUCTION OF GENOMIC LIBRARY The basic steps are as follows : Isolation of target DNA Fragmentation of DNA Vector preparation Ligation and introduction into host Storage of genomic library Screening of genomic library

6. 1. Isolation of genomic DNA

7. 2. Fragmentation of DNA a ) Mechanical shearing – using sonication or nebulizer b) Nuclease treatment – using Restriction Endonucleases Fragments treated with phosphatase – to remove terminal phosphate Sau3A Consists four-base-pair recognition site partial digestion

8. 3 . Vector preparation 4. Ligation and introduction into host 5. Storage of genomic library 6. Screening of genomic library a) Screening by colony hybridization

9. B) Screening by plaque hybridization

10. Applications of Genomic Library Genomic library construction is the first step in any DNA sequencing projects Genomic library helps in identification of novel pharmaceutically important genes. Helps in identification of new genes which were silent in the host. It helps in understanding of complexity of genomes. Can serve as a source of genomic sequence for generation of transgenic animals To study genetic mutations in cancer tissues

11. Hierarchical sequencing - This sequencing was used for the Human Genome Project

12. Genome wide association study(GWA Study)  A  genome-wide association study  ( GWA study , or  GWAS ), also known as  whole genome association study  ( WGA study , or  WGAS ). An   observational study  of a genome-wide set of  genetic variants  in different individuals . To see if any variant is associated with a trait. GWASs typically focus on associations between  single-nucleotide polymorphisms (SNPs) and traits like major human diseases . The first successful GWAS published in 2002 studied myocardial infarction . This study design was then implemented in the landmark GWA 2005 study investigating patients with  age-related macular degeneration , and found two SNPs with significantly altered  allele frequency  compared to healthy controls . The international HapMap Project is also based on this data.

13. cDNA Library cDNA Library - collection of cloned, restriction enzyme digested cDNA fragments where the cDNA represents all of the mRNA present at a particular stage in an organism or else all the genes that are expressed in the cells at different stages of its development. Steps in the construction of cDNA Library : 1. Isolation of mRNA 2. Preparation of complementary DNA fragments 3. cloning in suitable vector system 4. Transformation in suitable host . 5. Screening of cDNA

14. 1. Isolation of mRNA Lysis of cell using lysis buffer containing detergent Centrifuge the mixture and collect the supernatant Pass the mixture for column chromatography

15. 2. Preparation of cDNA Fragments Multiple approaches have been developed to prepare complementary DNA (cDNA) from isolated mRNA. In all approaches the 3 steps are performed : 1. First strand synthesis with a reverse transcriptase. 2. Removal of RNA template 3. Second strand synthesis

16. 3. cloning in suitable vector system The cDNA is ligated into the suitable vector to generate clone 4. Transformation in suitable host . Post ligation, clones are transformed in a suitable host to get colonies.

17. 5. Screening of cDNA A) Screening of specific cDNA plasmid in cDNA library using an antibody probe B ) Screening using radiolabelled probe

18. C) Screening using colony and plaque hybridization

19. Applications of cDNA Library 1. Discovery of novel genes. 2. Cloning of full-length cDNA molecules for in vitro study of gene function. 3.  Isolating cDNAs allows to use the cDNA to develop expression vectors so proteins of interest can be produced in high quantities, greatly simplifying the task of protein purification. (The human insulin gene or the corresponding cDNA has been inserted into the early region of a simian virus 40 vector in which all SV40 splice junctions were deleted while the early promoter and polyadenylation regions remained intact. The expression of insulin-coding sequences was tested in permissive monkey COS cells.) 4.  A great deal of information can be deduced about the possible structure and/or function of the protein encoded by a known cDNA

20. 5 . Knowing the cDNA sequence of a protein will frequently facilitate the development of antibodies and monoclonal antibodies. 6. cDNA sequence can be used as a probe to screen genomic libraries and isolate the gene encoding a particular cDNA

21. REFERENCES Gene Cloning and DNA analysis – T.A. Brown (6 th Edition ) Molecular Biology – by H.D. Kumar ( 2 nd revised Edition ) https://www.sciencedirect.com/topics/medicine-and-dentistry/gene-library https://ghr.nlm.nih.gov/primer/hgp/genome Fundamentals of Genetics – Peter J. Russell (2 nd Edition) https://nptel.ac.in/content/storage2/courses/102103045/download/mod3.pdf https://en.wikipedia.org/wiki/Genomic_library https://en.wikipedia.org/wiki/Genome-wide_association_study

22. THANK YOU