Genomic library

Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.

1. Welcome

2. GENOMIC LIBRARY Lulu S Kumar

3. Introduction In humans, approximately 25,000 genes exit among the 3 billion base pairs of DNA in the genome. To study anyone of these genes, a researcher first isolates it from all of the other genes in an organisms DNA. One isolation method has a relatively long history and involves the construction of a DNA library When a gene is identified and copied, it is said to have been “cloned”

4. DNA LIBRARY The term “ library ” can refer to a population of organism, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules. Collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study. For ease of purification, storage and analysis.

5. Two types of DNA Library Genomic library ( made from genomic DNA ) DNA Library cDNA Library ( made from cDNA - copy of mRNA )

6. For the construction of DNA Library Size of the gene Capacity of the vector Molecular tools Vectors

7. Size of the Library (ensure enough clones) Must contain a certain no: of recombinants for there to be a high probability of it containing any particular sequence. The formula to calculate the no: of recombinants: ln (1-P) N = ln (1-F) P: desired probability F: the fraction of the genome in one insert

8. Vectors for DNA Library

9. Genomic Library A library for me??? Why not?...You are the Backbone of Life……

10. What is Genomic L ibrary? Contains DNA fragments representing entire genome of an organism. Created using molecular cloning Genome size is expressed in terms of no: of base pairs. The sizes of genomes in different species are variable. There is a distinct difference in the genes of prokaryotes and eukaryotes. In prokaryotes, the structural genes coding for proteins are continuous while in eukaryotes, the coding regions ( exons ) are separated by non-coding regions ( introns ). Therefore, the construction of gene libraries for eukaryotes is more complicated.

11. Steps of Genomic Library construction Isolation of DNA from cells Digestion into small fragments Introduction into suitable vectors Insertion into bacteria DNA isolation Collection of Genomic DNA library

12. 1. Isolation of DNA ( purification) Eukaryotes : Prepare cell nuclei, remove proteins, lipids and other unwanted macromolecules by protease digestion and phase extraction. Prokaryotes : Extracted DNA directly from cells.

13. 2. Digestion into small fragments Physical shearing : Pipetting, mixing Restriction enzyme digestion : Partial digestion is preferred to get a greater lengths of DNA fragments.

14. Selection of restriction enzymes Ends produced (sticky or blunt) & the cleaved ends of the vector to be vector Whether the enzyme is inhibited by DNA modifications Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

15. 3. Introduction into suitable vectors Each fragment is different and have a unique DNA sequence Inserted into suitable vectors including plasmids and bacteriophage vectors. Vectors are digested with the same Restriction Enzymes and sealed to Human DNA using DNA Ligase enzyme . The resulting molecules are recombinant.

16. 4. Insertion into Bacteria Inserted into host bacteria ( E.coli ) The microbes are grown in culture. They are made to take up the DNA. They replicate their genome along with the vector genome contained with them. Produce clones of the original genome. This collection of clones which contains all the sequences found in the original source, including the sequence of interest forms the genomic library.

17. Multiplication and Production of clones Independent plasmid replication Host cell replication

18. Summary of Genomic Library construction

19. Screening of clones The methods used for the selection of recombinants include: Expression Screening or Direct S election of Recombinants Insertional Selection Inactivation M ethod Blue-White Selection M ethod Colony Hybridisation (Nucleic Acid Hybridisation) Technique

20. What are the uses of Genomic Library? Researchers can explore the genome of an organism to learn more about genomic structure and function They can map the genome, identifying the locations of specific genes. Helps to develop tests which can be used to locate genetic variations including mutations Useful in Recombinant DNA Technology, helps to genetically modify organisms and produce clones of desired types. Genomic library construction is the first step in any DNA sequencing projects. Genomic library helps in identification of the novel pharmaceutically important genes.

21. Shotgun sequencing Also known as shotgun cloning Originally used by Fred Sanger and his colleagues to sequence small genomes such as those of viruses and bacteria. DNA broken up into small fragments Sequenced using chain termination method to obtain reads Multiple overlapping reads of the target DNA are obtained Computer programmes use the overlapping ends of different reads to assemble them into a continuous sequence.

22. Random shotgun sequencing DNA isolated Cleaved into several fragments Randomly inserted into plasmids A) Some plasmids contain inserts which will be different from the others B) Some plasmids will have inserts which may contain some regions present in one insert and a few region present in other insert i.e. overlapping inserts. Overlapping sequences are identified using computer programmes which joins all sequences into one contiguous stretch.

23. Whole genome shotgun sequencing This includes 4 stages: Library construction :- A library of plasmid clones are constructed by transforming E.coli strains with plasmid. Random sequencing :- DNA is purified from plasmid. Thousands of DNA fragments are sequenced using automated sequencer and the whole genome is sequenced several times. Fragment alignment and gap closure :- Computer programmes assemble the sequenced fragments into longer stretches of sequence by comparing the overlapping sequence This ‘overlap comparison method’ resulted in a set of larger contiguous nucleotide sequence called contigs Contigs aligned in a proper order to form the completed genome sequence If there is gap between contigs , they are analysed and filled. Proof reading :- Ambiguities in the sequence can be resolved and mutations can be corrected.

24. Advantages and disadvantages of shotgun sequencing Advantages Faster process Uses only a fraction of DNA that clone-by-clone sequencing needs Particularly efficient if there is an existing reference sequence Less expensive than genetic mapping Disadvantages Requires vast amount of computing power and sophisticated software Errors can occur as genetic map is not used Reference genome is required for complete efficiency Repetitive genomes and sequences are difficult to be assembled

25. Reference Dr R. C. Dubey , A textbook of Biotechnology, 5 th edition, S. Chand Publishing, 2014, Chapter 6, Techniques of Genetic Engineering (Cloning Methods and DNA Analysis) Genomic Library, 98-131, Chapter 7, Genomics and Proteomics, 158-180 http:// www.sumanasinc.com/webcontent/animations/content/dnalibrary.html , Construction of Genomic Library, 2006 http:// www.slideshare.net/MariamNaseer/genomic-library , Genomic library, http:// www.slideshare.net/abdullahabobakr7/dna-library-lecturegene-libraries-and-screening , Genomic library, cDNA library and screening procedures http:// www.yourgenome.org/facts/what-is-shotgun-sequencing , shot gun sequencing, 2014 https:// en.wikipedia.org/wiki/Shotgun_sequencing , shotgun sequencing, 2015 https:// en.wikipedia.org/wiki/Genomic_library , genomic library, 2015