The Polymerase Chain Reaction (PCR) By Dr. NAGLAA FATHY Lecturer of Biochemistry & Molecular Biology Faculty of Medicine Benha University

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:

Invented by Kary Mullis Mullis and Faloona, Specific Mullis and Faloona, 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

Kary Mullis

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, 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, 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

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

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

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

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

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

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

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

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

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) = = 52 0 C Annealing T =T m 0 C -5

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

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

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

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

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

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

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

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

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 ).

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.

Basic reaction Basic reaction  Oligonucleotide primers  Design to flank the desired sequence  Steps include: ( 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

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

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

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).

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

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

In situ PCR

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.

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

Dr. Naglaa Fathy