Molecular diagnostic techniques allow for the accurate analysis of DNA, RNA, and proteins involved in various medical conditions like cancers, infections, and genetic disorders. Polymerase chain reaction (PCR) is commonly used to amplify DNA sequences of interest. Real-time PCR detects amplification as it occurs using fluorescent reporters. Blotting techniques like Southern blotting identify DNA, Northern blotting analyzes RNA, and Western blotting examines proteins. Hybridization methods involve binding of a probe to a complementary DNA or RNA sequence, as seen in in situ hybridization and microarrays. These molecular tools provide high sensitivity and specificity for disease investigation and diagnosis.

1. R.T.VINODHINI
Virulence assays
UNIT 4

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

26. LABELLING
TECHNIQUES

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