1. Online Counselling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. OC-SBT093-U01-01 Introduction Programmes and Courses  SEP – SBT093 – CP1-UN 01 2

3. School of Science and Technology, Online Counselling Resource… Credits  Academic Inputs by Arun Punaji More. M.Sc. (Microbiology) Experience: 11 Years.  arunmesh@hotmail.com 3

4. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counsellor at each study centre should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counselling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam. 4

5. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Define the term “Genomic library” Describe the various methods of preparation of genomic library Describe uses of genomic library in genetic engineering 5

6. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.6 Introduction-1  Isolation of gene of interest is based upon the successful cloning that gene and its subsequent identification with the help of some selectable markers such as auxotrophic mutations or antibiotic resistance.  Many times it is practically difficult to isolate a single gene of our interest due to absence of suitable selectable markers as mentioned above. 6

7. School of Science and Technology, Online Counselling Resource… Introduction-2  In such case all the genes of a genome is cloned and then gene of interest is identified by a suitable radio-labeled DNA probe.  Thus a set of clones of all the genes from a genome can be prepared constituting the genome library.  In this module students shall learn about the different methods of preparation of genome library and isolation and identification of desired gene or genes from this library. 7© 2007, YCMOU. All Rights Reserved.

8. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.8 Genomic Library-1 Genomic library is defined as a set of clones of all the genes of a genome of a organism. The size of genomic library is determined by the size of DNA fragment that can be inserted into cloning vector. The size of genomic library or the number of clones in a genomic library can be determined by following formula: N = ln(1-P)/ln(1-a/b) 8

9. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.9 Genomic Library-2 In this formula i.e. N = ln(1-P)/ln(1-a/b) N = number of clone in a genomic library a = average size of DNA fragment. b = total size of genome. p = probability of presence of any gene. 9

10. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.10 Genomic Library-3  Preparation of genomic library involve following steps: Purification of total cell DNA Partial digestion by restriction enzymes to produce DNA fragments. Cloning or insertions of these DNA fragments into suitable vectors such as lambda replacement vector, a cosmid, bacterial artificial chromosome or yeast artificial chromosome. Isolation of recombinant clones carrying separated gene of the genome; this is genomic library. 10

11. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.11 Genomic Library-4  Purification of total cell DNA:- Purification of total DNA is the first step in preparation of genomic library. To obtain total cell DNA in pure form one has to obtain  Pure cell culture and harvestation cell mass  Preparation of cell free extract  Removing the other cell ingredients to obtain DNA in pure form 11

12. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.12 Genomic Library-5  Purification of total cell DNA:- After growing and harvesting the pure culture; the cell mass is subjected to various techniques to obtain the cell free extract which contain all the cell content from which DNA is purified. First cell free extract is treated with protease enzymes such as pronase and proteinase K to break down proteins into smaller polypeptide units so that they can be easily precipitated out by phenol and chloroform treatment. 12

13. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.13 Genomic Library-6  Purification of total cell DNA:-  Next this enzyme treated cell free extract is deproteinized by mixing with phenol solution or phenol and chloroform mixture in 1:1 ratio; the proteins get precipitated out leaving the DNA and RNA in the aqueous solution.  Now to obtain the DNA free from RNA in the aqueous solution, RNA is degraded by enzyme ribonuclease into ribonucleotide subunits.  DNA is purified further by ultracentrifugation and concentrated by the ethanol precipitation at -20˚C.  DNA can also be purified by ion-exchange chromatography. 13

14. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.14 Genomic Library-7  Preparation of DNA fragments for cloning:- The purified genomic DNA is then partially digested with restriction endonucleases. For partial digestion of genomic DNA, only those restriction endonucleases are used which cut DNA molecules in very precise and reproducible manner. Some of the widely used restriction endonucleases are  EcoRI  BamHI  HindIII  TaqI 14

15. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.15 Genomic Library-8  Preparation of genome library Figure shows steps involved in Genomic library. 15

16. School of Science and Technology, Online Counselling Resource… Genomic Library-9  Insertion of DNA fragments into vectors:- Once number of fragments are obtained by restriction endonucleases digestion, these fragments are then inserted into suitable vectors such as:  λ replacement vector  Cosmid  Bacterial artificial chromosome (BAC) or P1 vector  Yeast artificial chromosome (YAC) 16© 2007, YCMOU. All Rights Reserved.

17. School of Science and Technology, Online Counselling Resource… Genomic Library-10  Isolation of recombinant clones:- The chimeric vectors with inserted genes are allowed to infect their suitable host bacteria which are grown on a suitable medium. A isolated pure colony of bacteria represent a clone of a isolated gene from the genome. All the colonies thus obtained represent the set of all genes from the genome and therefore a library of the genome. The library of these genes are then searched for a desired gene or genes by radio labeled DNA probe using DNA–DNA hybridization technique. 17© 2007, YCMOU. All Rights Reserved.

18. School of Science and Technology, Online Counselling Resource… Genomic Library-11 cDNA Library:- Preparation of cDNA Library using mRNA cDNA library make isolation of a desired gene easy and less tedious involving less steps than involved in genome library. 18© 2007, YCMOU. All Rights Reserved.

19. School of Science and Technology, Online Counselling Resource… Genomic Library-12 Probing of genomic library:- – Plaque and colony hybridization probing – Abundancy probing on cDNA library – Heterologous probing – Probing with oligonucleotide probes 19© 2007, YCMOU. All Rights Reserved.

20. School of Science and Technology, Online Counselling Resource… © 2007, YCMOU. All Rights Reserved.20 Genomic Library-13 Plaque and colony hybridization probing:- – Recombinant DNA is detected directly in bacterial colonies and bacteriophage plaques. – The colonies or plaques are transfered onto nitrocellulose papers and treated to denature the DNA molecules and allowed to react with radio-labeled probes. – The membrane is then autoradiographed and position of colony or plaque containing the desired recombinant DNA is fixed and isolated in pure form for further study. 20

21. School of Science and Technology, Online Counselling Resource… Genomic Library-14 Abundancy probing on cDNA library:- – Individual clones from the cDNA library itself is used as probe to screen the library. – The clone which hybridizes with maximum clones in the library is the desired clone present in the cDNA library. 21© 2007, YCMOU. All Rights Reserved.

22. School of Science and Technology, Online Counselling Resource… Genomic Library-15 Heterologous probing:- – Uses known gene from one species to identify the same gene in another related species by the DNA hybridization technique. – The heterologous probing uses the principle that there is nucleotide similarity between two genes encoding for the same protein in two different organisms due to conservation of gene structure during evolution. 22© 2007, YCMOU. All Rights Reserved.

23. School of Science and Technology, Online Counselling Resource… Genomic Library-16 Probing with oligonucleotide probe:- – The nucleotide sequence of a gene whose protein has been sequenced can be deciphered and used to construct a DNA fragment chemically in laboratory. – This chemically synthesized DNA fragment of known nucleotide sequence is called as oligonucleotide probe. 23© 2007, YCMOU. All Rights Reserved.

24. School of Science and Technology, Online Counselling Resource… What We Learn…..  Definition of genomic library  Methods of preparation of genomic library.  Principle of preparation of genomic library.  Technique of identification of the clone containing desired gene. 24© 2007, YCMOU. All Rights Reserved.

25. School of Science and Technology, Online Counselling Resource… Critical Thinking Question Why genomic library is constructed usually for less complex genome of microorganisms? 25© 2007, YCMOU. All Rights Reserved.

26. School of Science and Technology, Online Counselling Resource… Tips for Critical Thinking Question Manageable size of simple genome Produce less clones that can be analyze easily. 26© 2007, YCMOU. All Rights Reserved.

27. School of Science and Technology, Online Counselling Resource… Study Tips Book: Title: Molecular Biology Of The Gene Author: J. D. Watson, Becker, Bell, etc Publication: Pearson Education Book: Title: Gene Cloning and DNA analysis – An Introduction Author: T. A. Brown Publication: Blackwell Publishing 27© 2007, YCMOU. All Rights Reserved.

28. End of the Presentation Thank You ! 28