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

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

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

6. Kary Mullis

7. 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. 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. Progress was limited by primer synthesis and polymerase purification issues. Mullis properly exploited amplification Mullis properly exploited amplification

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

9. Amplify DNA PCR  I I I In vitro amplification (in a test tube)  E E E Enzymatic: Taq polymerase – Temperature-resistant DNA polymerase ( Thermus aquaticus) HHHHeat resistant BBBBest for <2 kb target

10. Takes advantage of basic requirements of replication  A DNA template  Nucleotides  Primers  polymerase PCR is DNA replication in a test tube

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

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

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

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

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

16. Melting temperature T m o C :Temperature at which T m o C :Temperature at which half possible H bonds are half possible H bonds are formed. formed. T m o C = 2(A+T) + 4(G+C) 5 / - AGACTCAGAGAGAACCC-3 / 4Gs 5Cs 7As 1T T m o C= (4x9) + (2x8) = 36+16 = 52 0 C Annealing T =T m 0 C -5

17. Heat-stable polymerase is vital to the ease of the process…

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

19. The Thermus aquaticus DNA polymerase Taq Taq  Not permanently destroyed at 94ºC  Optimal temperature is 72ºC

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

21. 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

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

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

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

25. How many copies? No target products are made until the third cycle. No target products are made until the third cycle. The accumulation is not strictly a doubling The accumulation is not strictly a doubling at each cycle in the early phase. At 30 cycles there are 1,073,741,764 target At 30 cycles there are 1,073,741,764 target copies (~1×10 9 ).

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

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

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

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

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

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

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

35. In situ PCR

36. 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.

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

38. Dr. Naglaa Fathy