بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. The Polymerase ChainReaction (PCR) By Dr. NAGLAA FATHY Lecturer of Biochemistry & Molecular Biology Faculty of Medicine Benha University E-mail : naglaa_fathy722000@yahoo.com Nagla.alhusseini@fmed.bu.edu.eg

3. What is the PolymeraseChain Reaction? • It’s a means of selectively amplifying a particular segment of DNA. • The segment may represent a small part of a large and complex mixture of DNAs:

4. Invented by Kary Mullis Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Nobel Prize 1993

5. Kary Mullis

6. Did He Really Invent PCR? • The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971:– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346. • Progress was limited by primer synthesis and polymerase purification issues. • Mullis properly exploited amplification

7. PCR Specifically targets and amplifies a SINGLE sequence from within a complex mixture of DNA. • How is this different from cloning?

8. Amplify DNAPCR • In vitro amplification (in a test tube) • Enzymatic: Taq polymerase – Temperature-resistant DNA polymerase ( Thermus aquaticus) • Heat resistant • Best for <2 kb target

9. Takes advantage of basicrequirements of replication • A DNA template • Nucleotides • Primers • polymerase PCR is DNA replication in a test tube

10. PRIMERS Primers: short ssDNA sequences complementary to border of sequence of interest

11. Primers Must have some information about sequence flanking your target Primers provide specificity

12. Primers • ends pointing towards each other • Complementary to opposite strands with 3’ • Should have similar melting temperatures

13. PCR Region of interest: between primers 2. Anneal 3. Extend Taq polymerase: enzymatic extension

14. PCR Repeated Cycles of 1. Denaturation 2. Annealing 3. Extention 1.Denaturation. 2. Anneal 3. Extend

15. Melting temperature TmoC :Temperature at which half possible H bonds are formed. TmoC = 2(A+T) + 4(G+C) 5/ - AGACTCAGAGAGAACCC-3/ 4Gs 5Cs 7As 1T TmoC= (4x9) + (2x8) = 36+16 = 520C Annealing T =Tm0C -5

16. Heat-stable polymerase is vitalto the ease of the process…

17. Thermus aquaticus Thermus aquaticus from hot springs in Yellowstone National Park, USA.

18. Taq Not permanently destroyed at 94ºC Optimal temperature is 72ºC The Thermusaquaticus DNApolymerase

19. Problems with Taq Taq DNA polymerase - thermostable • Lack of 3′-5′ exonuclease – proofreading ►Error rate = 2 × 10-4 nucleotdes/cycle • Newer polymerases have high fidelity High fidelity polymerase - HiFi Taq

20. Termplates for PCR • Small amount of template • In theory a single molecule • Do not need to isolate sequence of interest • DNA template need not be highly purified • DNA is stable in absence of nucleases

21. Templates for PCR 􀂄 Dried blood 􀂄 Semen stains 􀂄 Vaginal swabs 􀂄 Single hair 􀂄 Finger nail scrapings 􀂄 Egyptian mummies 􀂄 Buccal Swab 􀂄 Tooth brushes

22. Basic reaction ►Thermocycling, PCR machine 􀂃 Previously – need to overlay oil to prevent evaporation 􀂃 Automatically Change temperature 􀂃 Temperature gradient

23. • 30–35 cycles each comprising: – denaturation (95°C), 30 sec. – annealing (55–60°C), 30 sec. – extension (72°C), time depends on product size The Basics of PCR Cycling

24. How many copies? • No target products are made until the third cycle. • The accumulation is not strictly a doubling at each cycle in the early phase. • At 30 cycles there are 1,073,741,764 target copies (~1×109).

25. • Increasing the cycle number above ~35 has little positive effect. • The plateau occurs when: – The reagents are depleted – The products re-anneal – The polymerase is damaged - Unwanted products accumulate. How many cycles?

26. Basic reaction • Oligonucleotide primers • Design to flank the desired sequence • Steps include: (30-40 steps) 􀂃 Denaturation at 94°C 􀂃 Primer annealing at Tm-5°C 􀂃 Extension at 72°C

28. Reverse Trascription PCR (RT-PCR) Use mRNA as a template Total cellular RNA - faster Contamination of genomic DNA – false result Primer specific to exons Treat sample with DNase Can be quantitative rtPCR

29. Multiplex PCR • Simultaneously modification of more than one locus in the same reaction • Rapid and convenient – screening • Included different set of primers

31. Quantitative or Real Time PCR • Monitors the fluorescence emitted during the reaction as an indicator of amplicon production • during each PCR cycle. 􀂃 The parameter CT (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold (background).

32. Quantitative or Real Time PCR Three different fluorescence systems: ►Hydrolysis probes ►Hybridizing probes ►DNA binding agents SYBR-Green I

33. Molecular Beacons • Uses FRET • Fuorescence Resonance Energy Transfer • Uses two sequence specific oligonucleotides labeled with fluorescent dyes

34. In situ PCR

35. Applications of PCR • Mutation detection . • Diagnosis or screening of acquired diseases,: e.g. AIDS, HBV & HCV. • Prenatal diagnosis • DNA profiling in forensic science • Quantitation of mRNA in cells or tissues.

36. Problems with PCR • Contamination • Theoretically one molecule can amplify • Takes one mismatch early on to amplify the wrong fragment

37. Thank you Dr. Naglaa Fathy