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

2. OC-SBT093-U01-03 Introduction Programmes and Courses  SEP – SBT093 – CP1- UN01

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

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.

5. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives After studying this modules students should be able to Describe various nucleic acid probes used in genetic engineering Describe preparation of nucleic acid probes Describe the applications of nucleic acid probes

6. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Introduction-1  In genetic engineering after the cloning genes from a cell, the desired gene has to be identified from a set of population of isolated genes.  The desired gene is identified using a DNA probe or also called as molecular probe which specifically binds to the desired nucleotide sequences.  The identification of desired gene by molecular probe is based upon the DNA-DNA hybridization. Students shall learn about these DNA probes in this module.

7. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 Molecular Probes-1  DNA Probes:- Molecular probes are segments of DNA or RNA of known nucleotide sequences. They are prepared by purifying the DNA or RNA fragments or synthesized chemically. DNA probe is used to search for a desired nucleotide sequence. When this probe is mixed with mixture of DNA molecules, it forms stable hybrid DNA molecule with its complementary DNA strands. The mixture of DNA molecules can be either genomic library distributed in different colonies of clones or separated DNA molecules on a gel by electrophoresis.

8. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 Molecular Probes-2 Isolation and purification of DNA probes:- cDNA gene can be used as DNA probe. cDNA is selected from cDNA clone library,labeled and used to probe the remaining clones in the library. This probing is repeated with number of cDNA probes from the same library until a probe is selected which hybridizes with large number of clones in the library. This cDNA probe which hybridizes with large number of cDNA clones in the library represent the mRNA present in large number in the original sample.

9. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 Molecular Probes-3 Oligonucleotide probes:- This is synthetic nucleotide sequences up to 150 nucleotides prepared in lab. Oligonucleotide probe is used in cases where amino acid sequences of desired gene product is known. Since except methionine and tryptophan, all amino acids are coded by at least two codons, different oligonucleotide probes are prepared in different possible sequences representing the different codons for the amino acid sequence in the protein. One of these sequence of the oligonucleotide shall be correct probe which will correctly hybridize with the desired gene.

10. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 Molecular Probes-4 Heterologous probes:- Gene coding for the same protein in different living forms show conservation in their structures. Therefore gene from one living form can be used as DNA probe to identify a gene coding for the same protein in another living form.

11. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Molecular Probes-5  RNA probes:- – Unlike the DNA probe which can hybridizes with both DNA and RNA, RNA probe can hybridizes only with RNA. – Unlike the DNA probe, the RNA probes are single strands. – RNA probe is used to detect the mRNA level in order to judge the expression of desired gene. – RNA probe is widely used in rRNA based molecular phylogenetic.

12. School of Science and Technology, Online Counseling Resource… Molecular Probe-6 RNA Probe:- – RNA probes are used in the diagnosis of number of viral infections by detecting their RNAs.

13. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Molecular Probes-7  Labeling of DNA probes:- – To label radioactively, DNA probe is synthesized in the presence of radio-labeled precursors. – Probe can also be labeled by adding the radio- labeled molecule at the end of intact DNA probe molecule. – Enzyme poly-nucleotide kinase adds γ- phosphate from ATP to the 5'OH group of the DNA probe molecule. – DNA precursors are either labeled with radio- active atom(such as 32 P) or with fluorescent moiety. a

14. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 Molecular Probes-8  Labeling of DNA probes:- Fig shows Labeling of DNA probe With Radio-isotope 32 P

15. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 Molecular Probes-9  Labeling of DNA probes:-

16. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 Molecular Probes-10  Applications of molecular probes:- DNA probes are used to isolated and identify the desired gene or DNA fragment of known nucleotide sequences. RNA probes are used to desired gene or DNA fragment for a particular nucleotide sequence. The small chemically synthesized DNA probes are used  To introduce site directed mutation to produce restriction sites into clones DNA fragment.  As primer in DNA elongation in polymerase chain reaction process.

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 Applications of DNA Probes DNA probe is also used to determined the amount and size of DNA and mRNA molecules in the mixed population of DNA and mRNA molecules.

18. School of Science and Technology, Online Counseling Resource… What We Learn…..  Preparation of DNA probes.  Preparation of RNA probes.  Labeling of DNA and RNA probes.  Uses of DNA and RNA probes. © 2007, YCMOU. All Rights Reserved.18

19. School of Science and Technology, Online Counseling Resource… Critical Thinking Question  Why chemically synthesized DNA probes are prepared of only below 30bp size? 19© 2007, YCMOU. All Rights Reserved.

20. School of Science and Technology, Online Counseling Resource… Tips for Critical Thinking Question  Error in nucleotide sequences. © 2007, YCMOU. All Rights Reserved.20

21. 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 © 2007, YCMOU. All Rights Reserved.21

22. End of the Presentation Thank You ! © 2007, YCMOU. All Rights Reserved. 22