SIMPLE TO UNDERSTAND

1. Polymerase Chain Reaction (PCR) and Its Applications by Dr. B. K. Iyer

5. What is PCR? : The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg ++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

6. The Reaction THERMOCYCLER PCR tube

7. The steps Denature (heat to 95 o C) Lower temperature to 56 o C Anneal with primers Increase temperature to 72 o C DNA polymerase + dNTPs

8. The steps

10. The outcome

14. Molecular identification

15. Types of PCR

16. Detection of Unknown Mutations Molecular Identification:

17. Detection of Unknown Mutations SSCP gels: “shifts” representing a mutation in the amplified DNA fragment

18. Classification of Organisms 1) Relating to each other 2) Similarities 3) Differences * Fossils * Trace amounts * Small organisms ! DNA ! Molecular Identification: Insufficient data

19. Classification of Organisms

20. Classification of Organisms Rademaker et al. 2001

21. Detection Of Pathogens Molecular Identification:

22. Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al .1994) Molecular Identification:

23. Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al .1994)

24. Genotyping by STR markers Molecular Identification:

26. Sequencing Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! Fragments of DNA differing only by one nucleotide are generated Nucleotides are either or radioactive fluorescent

27. Sequencing

28. Classical Sequencing Gel Sequencing:

29. Reading Classical Sequencing Gels Sequencing:

30. Reading Classical Sequencing Gels Sequencing:

31. Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing