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

2. OC-SBT093-U01-04 Introduction Programmes and Courses  SEP – SBT093 – CP1 - UN01

3. School of Science and Technology, Online Counseling Resource… Credits Academic Inputs by – ARUN PUNAJI MORE QUALIFICATION: M.Sc. (Microbiology) Experience: 11 years in academic field

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 module students should be able to  Describe polymerase chain reaction(PCR)  Describe different applications of PCR  Describe the automated gene sequencing

6. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Introduction Structure of gene is studied by finding out its nucleotide sequences; this is called as gene sequencing. If we know the structure of a gene, we can use that gene as a probe to identify the presence or absence of that gene in a genetic disease involving that gene. Gene sequencing and gene amplifications are independently or in combinations are widely used by molecular biologists for variety of applications in genetic engineering and biotechnology; in this modules students shall learn the same.

7. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 Polymerase Chain Reaction-1 PCR is nucleic acid synthesis reaction by which a desired gene is amplified by synthesizing its millions of copies. PCR involve following steps: Synthesis of a oligonucleotide primer complementary to sequences at the border of the gene to be amplified Hybridization of this primer to the desired gene Synthesis of new DNA fragment in the presence of special DNA polymerase I enzyme called as Taq polymerase from microorganism Thermus acquaticus.

8. School of Science and Technology, Online Counseling Resource… Polymerase Chain Reaction-2 Principle of PCR:-

9. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 Polymerase Chain Reaction-3 The oligonucleotide primer should not be too long or too short for the successful PCR. A short primer give amplification of different fragments of DNA molecule. Long primer slow down the rate of hybridization reaction prolonging the PCR reaction. DNA fragment to be amplified should be less than 3kb; ideally less than 1kb. DNA fragment more that 3kb up to 40kb requires special method for its amplification.

10. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 Polymerase Chain Reaction-4 PCR cycle consist of Denaturation Hybridization of DNA template and primer Synthesis of new DNA strand The DNA molecule containing the gene to be amplified is first denatured at 94 ° C to single stranded DNA molecules. The reaction mixture then cooled down to annealing temperature required for hybridization of primer and DNA template.

11. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Polymerase Chain Reaction-5 The primer and DNA template annealing temperature is selected 1-2 ° C less that of the melting temperature of primer and DNA template. The melting temperature (Tm) of primer and DNA template is calculated by the following formula: Tm = [(4x(G+C))+(2X(A+T))] [G+C] is the number of G and C nucleotides. [A+T] is the number of A and T nucleotides.

12. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 Automated Gene Sequencing-1 Automated gene sequencing is done by chain termination method. However there is one difference between the traditional chain termination method and chain termination method used in automated gene sequencing. In traditional chain termination method, DNA probe is labeled with radio-active molecule or fluorescent moiety. In automated gene sequencing, DNA probe is labeled only with fluorescent moiety.

13. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Automated Gene Sequencing-2 A different fluorochrome is used for each of the four dideoxynucleotide. The chain termination reaction is carried out in a single tube. Different four fluorochrome produce different fluorescent signals which are detected by photosensitive instrument attached to a computer. The computer process the signals detected by photosensitive detector and convert it into appropriate sequence.

14. School of Science and Technology, Online Counseling Resource… Automated Gene Sequencing-3 Comparison of manual gene sequencing With gel electrophoresis and automated gene Gene sequencing DNA sequence trace chromatogram Generated by gene sequencer © 2007, YCMOU. All Rights Reserved.14

15. School of Science and Technology, Online Counseling Resource… Automated Gene Sequencing-4 Automated gene sequencer is much faster than manual gene sequencing. Automated gene sequencer reads 96 different sequences within two hours. Automated gene sequencing is not tedious as manual gene sequencing. © 2007, YCMOU. All Rights Reserved.15

16. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 What We Learn... Principle of polymerase chain reaction Applications of polymerase chain reaction Limitations of PCR in gene sequencing. Automated gene sequencing Advantages of automated gene sequencing

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 Critical Thinking Question Why special DNA polymerase is used for gene sequencing method?

18. School of Science and Technology, Online Counseling Resource… Tips For Critical Thinking Question Dual functions of DNA polymerase gives errors in DNA sequencing.

19. School of Science and Technology, Online Counseling Resource… Study Tips Book: Title: Molecular Biology Of The Gene Author: J. D. Watson, Baker, Bell, etc Publication: Pearson publication Book: Title: Gene Cloning and DNA Analysis – An introduction Author: T. A. Brown Publication: Blackwell Publishing

20. End of the Presentation Thank You !