Genomic library construction using lambda. phagepptx

Genomic library construction using lambda phage.pptx

1. 18 CONSTRUCTION OF GENOMIC LIBRARY USING LAMBDA PHAGE

2. Gene libraries Genomic library: Consist of total chromosomal DNA of an organism. cDNA library: It is a collection of DNA molecules that are synthesized from the mRNA (messenger RNA) molecules present in a specific cell type or tissue at a particular point in time. Gene libraries serve as valuable resources for molecular biology research, enabling the study of genes, genetic elements, and genome organization. Two types Genomic library cDNA library A large collection of DNA fragments cloned from a given organism, tissue, organ, or cell type 19

3. Purpose of Genomic Libraries Study Genetic Diversity Gene Mapping and Localization Understand Genetic Disorders Facilitate Genome Sequencing Projects Purpose of cDNA Libraries Study Gene Expression Identify mRNA Transcripts Analyze Alternative Splicing Investigate Tissue-Specific Expression 20

4. How it started? The researchers in 1970 were curious to study and understand the genetic material of organisms comprehensively. The development of recombinant DNA technology, enabling the manipulation of DNA fragments from diverse sources was also a driving factor for the creation of gene libraries. 22

5. Construction of genomic library using lambda phage Lambda phage is a bacteriophage that infects E.coli bacteria. Advantages Accommodates large DNA inserts, easy manipulation, and efficient packaging of recombinant DNA into phage particles. Phage lambda system 23

6. Steps in Constructing a Genomic Library Isolation of Genomic DNA Digestion with Restriction Enzymes Ligation into Lambda Vector Insertion of DNA Fragments into Vector Packaging into Lambda Phage Particles Infection of Host Bacteria Screening for Recombinant Clones Isolation of Recombinant Clones Analysis of Cloned DNA Fragments 24

7. Isolation of Genomic DNA It can vary depending on the organism under study. It could be tissue samples, blood, cultured cells, or even whole organisms. Mechanical disruption (e.g., grinding or homogenization), enzymatic digestion (e.g., using lysozyme or proteinase K), or chemical lysis (e.g., using detergents like SDS or Triton X-100). Components like proteins, lipids, and RNA need to be removed to isolate the DNA. This is done through a series of purification steps. (protease treatment, phenol- chloroform extraction ) Selection of Source Material: Cell lysis Removal of Cellular Components 25

8. Isolation of Genomic DNA DNA can be precipitated out using ethanol or isopropanol. Adding a high concentration of alcohol to the DNA solution, causing the DNA molecules to come out of solution and form a visible pellet at the bottom of the tube. The DNA pellet is washed with a buffer to remove residual contaminants and alcohol. It is then resuspended in a buffer solution, such as TE buffer (Tris-EDTA buffer), which helps maintain the stability of the DNA. DNA Precipitation Washing and Resuspension 26

9. Isolation of Genomic DNA The quality and quantity of DNA can be done using various methods, such as UV spectrophotometry to measure DNA concentration and assess purity, agarose gel electrophoresis to check for DNA integrity and size distribution, and fluorescence-based assays (e.g., Qubit fluorometer) for accurate quantification . T he isolated genomic DNA is typically stored at -20°C or -80°C to maintain its stability and integrity for future use in molecular biology experiments, including the construction of genomic DNA libraries. Quality Assessment Storage 27

10. Digestion with Restriction Enzymes Genomic DNA is digested using restriction enzymes to generate a collection of DNA fragments. Enzymes are chosen based on specific recognition sequences and cutting sites within the genomic DNA Very large genomes such as human DNA, are digested with enzymes that produce long DNA fragments. 28

11. Ligation into Lambda Vector The DNA fragments created by restriction digestion with sticky ends can be combined with any other DNA fragments cut with the same restriction enzyme. Modified lambda phage genome with sites for insertion of foreign DNA fragments are used. DNA fragments from the genomic library are ligated into the lambda vector using DNA ligase. 29

12. Active site for cos insertion in genomic library construction In phage vector-based genomic library construction, DNA fragments are inserted at the cos site. The cos site is a short, specific DNA sequence recognized by the lambda phage. CosN : This region contains the " cosN " sequence, which is typically composed of 12 nucleotides. The sequence is 5'-GCTCTTCCTTTC-3' in lambda phage DNA. This is recognized by the lambda phage terminase enzyme during the packaging process. The terminase enzyme recognizes and binds to the cosN sequence to initiate packaging of the DNA fragment into the lambda phage head. 30

13. Active site for cos insertion in genomic library construction CosQ : This region contains the " cosQ " sequence, which serves as the site where DNA cleavage occurs during packaging, it is typically composed of 12 nucleotides. The sequence is complementary to cosN and is 5'-GAAAGGAAGAGC-3' in lambda phage DNA. The terminase enzyme cleaves the DNA at the cosQ site to generate cohesive (sticky) ends, which are necessary for the insertion of the DNA fragment into the vector. The cosmid vector's complementary cos site enables it to be packaged into lambda phage particles with inserted DNA fragments, creating recombinant lambda phage particles used for bacterial host infection in library construction. 31

14. Infection of Host Bacteria Typically Escherichia coli (E. coli) bacteria are used. Lambda phage particles containing recombinant DNA infect host bacteria, delivering the genomic library. Genomic libraries have been prepared from hundreds of different species. Many clones must be created to be confident that the genomic library contains the gene of interest. 32

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16. Screening for Recombinant Clones Selective Medium: Infected bacterial cells are plated onto agar plates containing a selective medium that allows only recombinant clones to grow. Identification: Recombinant clones are identified based on their ability to grow on the selective medium. 34

17. Screening for Recombinant Clones S creening by Colony Hybridization: Principally this screening technique involves hybridization between labelled DNA probe and a target DNA sequence. Therefore, a target gene sequence can be accurately identified. Polymerase Chain Reaction (PCR) If the sequence of the DNA fragment of interest is known, PCR can be used to amplify that specific sequence from the genomic library. Clones containing the amplified sequence are identified as recombinants. Functional Assays In some cases, the DNA fragment of interest may encode a functional gene or protein. Functional assays can be performed to screen for clones expressing the desired phenotype or protein activity 35

18. Characterization of Cloned DNA Fragments Once potential recombinant clones have been identified through screening methods, the cloned DNA fragments within these clones need to be characterized. This typically involves analyzing the size, structure, and integrity of the inserted DNA fragments. Sequencing: One of the most common methods used to characterize cloned DNA fragments is DNA sequencing. Sequencing allows researchers to determine the exact nucleotide sequence of the inserted DNA fragment, providing valuable information about the gene(s) or genetic elements contained within it. 36

19. Comparative Analysis The sequenced DNA fragments can be compared to known sequences in databases to identify genes, regulatory elements, or other functional elements. Comparative analysis helps researchers understand the biological significance of the cloned DNA fragments and their potential roles within the genome. Validation of Cloned DNA Fragments: To ensure the accuracy and reliability of the genomic library, the cloned DNA fragments need to be validated. This may involve various validation assays, such as PCR amplification using specific primers designed to target the cloned DNA fragment, or functional assays to confirm the activity or expression of genes contained within the fragment. 37

20. THANK YOU 39