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

2. OC-SBT093-U01-02 Introduction Programmes and Courses  SEP – SBT093 – CP1-UN 01 2

3. School of Science and Technology, Online Counseling 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 Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  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 Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Describe techniques of DNA hybridization Describe DNA-RNA hybridization Explain the applications of nucleic acid hybridization techniques 5

6. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Introduction-1 DNA molecule is double strands of nucleotides wound around each another in spiral fashion. These strands are held together by weak forces such as hydrogen bonds and can be reversibly separated from each another by heat treatment at a particular temperature. This properties of separation and re- association of two complementary strands of DNA molecules are widely employed in genetic engineering; in this module students shall learn about it 6

7. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 DNA-DNA Hybridization-1 The complementary DNA strands get separated from each another at higher temperature; these single strands can again re-associate with each another when temperature is lower. This re-association of complementary DNA strands at appropriate temperature and salt concentration is called as DNA- DNA hybridization. The single DNA strands can be from same genome or from different genome. 7

8. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 DNA-DNA Hybridization-2 In DNA-DNA hybridization formation of stable hybrid DNA molecules depends upon the homology of these strands. Homologous DNA strands form stable hybrid DNA molecule. In vitro DNA-DNA hybridization is carried out on nitrocellulose or nylon membrane, in electrophoresis gel or in aqueous solution. 8

9. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 DNA-DNA Hybridization-3 Principle of DNA hybridization:- 9

10. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 DNA-DNA Hybridization-4 DNA-DNA hybridization on membranes:- Bacterial colony or bacteriophage plaques are transferred to nitrocellulose or nylon membranes. Cell materials except DNA are removed. Incubation at 80 ° C for short time binds the DNA strands tightly to nitrocellulose membrane. If the DNA strands are transferred to nylon membrane then the DNA strands are bound by UV irradiation. 10

11. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 DNA-DNA Hybridization-5 DNA-DNA hybridization on colonies:- 11

12. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 DNA-DNA Hybridization-6 DNA-DNA hybridization on membranes:- The membrane bound DNA molecule is then identified by radio labeled DNA probe of known nucleotide sequences. the DNA probe get hybridized with the complementary DNA strand immobilized on the membrane. Since the DNA probe is radio labeled the position of hybrid strand and therefore the desired gene can be found out on the membrane by exposing the membrane to X- rays. 12

13. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 DNA-DNA Hybridization-7 Southern hybridization:- – It is DNA-DNA hybridization technique. – It involves the separation of DNA fragments on electrophoresis gel. – Transfer of separated DNA fragments onto nitrocellulose membrane – Treatment of this membrane to denature the adsorbed DNA fragments. – The desired DNA fragment is detected by radio-labeled DNA probe which hybridizes specifically to its complementary DNA strand. 13

14. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 RNA-RNA Hybridization-8 Northern hybridization:- The RNA-RNA hybridization is carried out by special technique known as Northern hybridization of Northern blotting Northern blotting technique isolate and identify the desired RNA from the mixture of number of RNA using RNA probe; the principle and the methodology is same as that of Southern blotting. Northern blotting is widely used to isolate and identify RNAs from RNA viruses in the diagnosis of infections caused by RNA viruses. 14

15. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 DNA-DNA Hybridization-9 DNA-DNA hybridization In liquid:- – DNA hybridization in liquid is used to quantitate genes and their RNA products. 15

16. School of Science and Technology, Online Counseling Resource… In Situ Hybridization Chromosome in situ hybridization:- – Probing of metaphase and prometaphase chromosome is carried out with the labeled DNA probe to locate the position of a desired gene on chromosomes Tissue in situ hybridization:- – Mounted slice of tissue is probed with RNA probe to detect the expression of mRNA in that tissue. – To study tissue specific expression of genes. © 2007, YCMOU. All Rights Reserved. 16

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 DNA-DNA Hybridization-10 Applications of Southern hybridization:- – The Southern hybridization is widely used to isolate a desired gene from DNA fragments. – The technique is also used to identify the desired gene from mixture of DNA fragments using specific DNA probe. – If one the know the specific gene unique to a particular bacterial pathogen or virus, then southern hybridization is used to identify such gene and therefore diagnosis of the infection caused by such pathogen. 17

18. School of Science and Technology, Online Counseling Resource… Southern Blotting DNA-DNA hybridization in diagnosis:- 18 © 2007, YCMOU. All Rights Reserved.

19. School of Science and Technology, Online Counseling Resource… DNA-DNA Hybridization-11 Applications of nucleic acid hybridization:- – Nucleic acid hybridization is used in Southern blot- to detect and isolate a desired gene Northern blot to detect gross expressions of a desired gene. In detection of mRNA in a particular type of tissue. Nucleic acid hybridization is used in RFLPs, VNTR and DNA fingerprinting technology. For detection of deletion type of mutation by restriction mapping. For in situ hybridization to detect the expression of mRNA. 19 © 2007, YCMOU. All Rights Reserved.

20. School of Science and Technology, Online Counseling Resource… What We Learn… Principles of nucleic acid hybridization Procedure of carrying out DNA-DNA hybridization Applications of nucleic acid hybridization 20 © 2007, YCMOU. All Rights Reserved.

21. School of Science and Technology, Online Counseling Resource… Critical Thinking Question How stability of hetero-duplexes formed in DNA-DNA hybridization is determined? 21 © 2007, YCMOU. All Rights Reserved.

22. School of Science and Technology, Online Counseling Resource… Hint For Critical Thinking Question DNA melting temperature (Tm) 22 © 2007, YCMOU. All Rights Reserved.

23. School of Science and Technology, Online Counseling 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 23 © 2007, YCMOU. All Rights Reserved.

24. End of the Presentation Thank You ! 24 © 2007, YCMOU. All Rights Reserved.