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virulence assays - molecular diagnostic techniques
- 2. • Virulence – degree of disease causing ability • VIRULENCE( MEDICAL TERMS) – SEVERITY OF A DISEASE Virulence
- 3. Molecular diagnostic techniques 🞄Analysis of DNA, RNA and Proteins 🞄HIGHACCURACY & REDUCED COST 🞄 Uses: oNeoplastic disorders oInfectious diseases oInherited conditions oIdentity determination
- 4. Types 🞄Amplification techniques: oPCR & its sub types 🞄 Blotting techniques: oSouthern blotting oNorthern blotting oWestern blotting 🞄Hybridization techniques: oIn situ hybridization oFISH oMicroarray
- 5. PCR 🞄 POLYMERASE CHAIN REACTION 🞄Amplification of DNA - large quantities of a specified DNA 🞄 Developed by Kary Mullis in 1984
- 6. PCR 🞄Step 1: Denaturation of DNA at 94- 98 °C
- 7. PCR 🞄Step 2:Annealing with primers at 48-56°C
- 8. PCR 🞄Step 3: Extension of primers at 68-72°C
- 10. PCR
- 11. Reverse Transcriptase- PCR 🞄 Amplifies DNA from RNA 🞄REVERSE TRANSCRIPTASE ENZYME 🞄 Taq/Tth polymerase – thermus thermophilus
- 14. Real time - PCR 🞄AMPLIFICATION DETECTED WHILE OCCURRING 🞄Data collection after each cycle – fluorescent reporter
- 15. Nested - PCR 🞄2 sets of primer – outer & inner 🞄Increases sensitivity & specificity 🞄1st round –Gene products – target for 2nd round 🞄2nd round – Primers binding site within 1st set
- 17. Blotting techniques 🞄ANALYTICALTECHNIQUE FORTHE IDENTIFICATION OF DNA, RNA OR PROTEIN 🞄 Southern blot – DNA 🞄 Northern blot – RNA 🞄 Western blot - Proteins
- 18. Southern blotting 🞄SIR EDWIN SOUTHERN - 1975 🞄To detect specific DNA sequence 🞄DNA digestion 🞄Gel electrophoresis 🞄Blotting 🞄Hybridization & washing 🞄Detection
- 20. Northern blotting 🞄JAMES ALWINE & GEORGE STARK – 1979 🞄To detect specific RNA sequence 🞄RNA isolation 🞄Gel electrophoresis 🞄Blotting 🞄Hybridization & washing 🞄Detection
- 22. Western blotting 🞄W Neal Burnette - 1981 🞄 IDENTIFICATION OF PROTEIN 🞄Antigen – protein of interest; Antibody – probe 🞄SDS PAGE 🞄Blotting 🞄Blocking 🞄Primary & secondary hybridization 🞄Detection 🞄Antigen – antibody interaction
- 24. Hybridization 🞄Formation of duplex between two complementary strands
- 25. In situ Hybridization 🞄Precise localization of a specific segment of target sequence within a histologic section 🞄DETECTEDTHROUGHCOMPLEMENTARY STRAND OFTARGETTO WHICHA REPORTER MOLECULE IS ATTACHED
- 27. FLUORESCE NCE IN SITU HYBRIDIZATI ON 🞄Fluorescent probes that bind to only those parts of a target sequence with a high degree of sequence complementarity
- 31. Microarray 🞄DNA chip (lab on a chip), next generation sequencing. 🞄Hybridization to detect the presence of target DNA/RNA 🞄Contain thousands of known immobilized DNA sequences. 🞄The fluorescently tagged sample to be sequenced is added. 🞄Genotyping – single stranded genomic DNA is added. 🞄Gene expression – cDNA is added.
- 33. Thank you