Pcr & dna microarray

PCR

1. POLYMERASE CHAIN REACTION & DNA MICROARRAY TECHNOLOGY Dr Ambika Jawalkar

2. POLYMERASE CHAIN REACTION Kary Mullis in 1983, Noble Prize in Chemistry in 1993 A scientific technique in Molecular Biology Amplification of a single or a few copies of DNA a cross several orders of magnitude Can generate thousands to millions of copies of a d esired DNA sequence within few minutes

3. PRINCIPLES OF PCR: Thermal cycling Selective & repeated amplification with help of Primers Taq Polymerase isolated from Thermus aquaticus As the reaction progresses the DNA generated is itself used as a template

4. PROCEDURE Mostly amplify DNA fragments up to 10kbs but some allow amplification of fragments up to 40kbs size Carried out in a reaction volume of 10-200µl in small reaction tubes of 0.2-0.5ml volumes Reaction tubes are placed in a thermal cycler

5. COMPONENTS DNA template / target DNA Primers Taq Polymerase dNTPs Buffer solution Magnesium Chloride salt solution

6. STEPS: Each cycle consists of 3 discrete temperature steps Denaturation step - @ 95ºc for 20 to 30 sec Annealing Step – 50 to 65ºc for 20 to 40 sec Extension / Elongation step

7. STAGES OF PCR Exponential amplification Leveling off stage Plateau

8. CLINICAL APPLICATIONS Role in diagnosis of Infectious diseases Role in Cancer diagnostics Genetic diseases & Paternity testing

9. VARIATIONS / MODIFICATIONS OF BASIC PCR TECHNIQUE: Reverse Transcription PCR (RT-PCR) Quantitative PCR (Q-PCR) Nested PCR Asymmetric PCR Multiplex PCR

10. DNA MICROARRAY TECHNOLOGY -The Diagnostics of Future Introduction: Central Dogma of Life DNA mRNA Protein Transcription Translation

11. This technology measures the activity of genes at a transcriptional level. The information that can be obtained by sequencing a gene is, Sequence of protein it encodes Can guess the function of the gene Can look for presence of mutations Can compare the gene sequence & the protein it encodes in different animal species Can study evolution of genes

12. S TEPS : Sample Preparation - isolation of total RNA - reverse transcription - labeling Hybridization - binding between the targets & probes - washing Detection - chip reading Data acquisition & analysis - collection & summary of raw data - statistical analysis of the data

13. DNA Microarrays / DNA chips : basic concept Small solid supports onto which the sequences from thousands of different genes are immobilized or a ttached at fixed locations. Are usually glass microscope slides or silicon chips or nylon membranes DNA is printed, spotted or synthesized directly on to the glass slide Each spot represents a particular gene sequence Spots can be DNA, cDNA or oligonucleotides

14. Dimensions of a gene chip

15. Principle: Hybridization Probing – a technique that uses fluorescently labeled nucleic acid molecules to identify complementary molecules

16. PROCEDURE

17. Interpretation of gene chip array

18. Types of Microarrays: 3 basic types of samples can be used to construct DNA microarrays Two are genomic Transcriptomic

19. Advantages: Follow activity of many genes at the same time Fast results Comparing the activity of many genes in diseased & healthy cells Categorize diseases into subgroups Limitations / Drawbacks: × too much data at once × results may be too complex to interpret × results are not always reproducible × still too expensive

20. Microarray applications (in brief) Expression analysis drug development, drug response & therapy development Mutation / Polymorphism analysis drug development, therapy development & tracking disease progression

21. CONCLUSION

22. THANK YOU