Polymerase Chain Reaction
(PCR)
Prepared by:
Paywast J. Jalal
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Molecular
Biology
History of polymerase chain reaction (PCR)
Definition and short technical overview of PCR
Applications of PCR
Practice the PCR
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Molecular Biology
Learning objectives
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The Problem...
There are a LOT of other sequences in a genome that we’re not
interested in detecting. (SPECIFICITY)
The amount of DNA in samples we’re interested in is VERY small.
(AMPLIFICATION)
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Two Big Problems:
Molecular Biology
How do we identify and detect a specific sequence in a
genome?
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• Kary B. Mullis developed the polymerase chain reaction
(PCR) in 1983.
• The simple process involved heating a vial containing the
DNA fragment to split the two strands of the DNA molecule,
adding oligonucleotide primers to bring about reproduction,
and finally using polymerase to replicate the DNA strands.
Each cycle doubles the amount of DNA, so multiple cycles
increase the amount of DNA exponentially, creating huge
numbers of copies of the DNA fragment.
• For his development of PCR, he was co-awarded the Nobel
Prize in chemistry in 1993.
Molecular Biology
PCR History
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Polymerase Chain Reaction
(PCR)
• PCR is a means to amplify a particular piece of DNA
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• PCR can make billions of copies of a target
sequence of DNA in a few hours
• PCR was invented in the 1984 as a way to make
numerous copies of DNA fragments in the
laboratory
• Its applications are vast and PCR is now an integral
part of Molecular Biology
Molecular Biology
• Amplify= making numerous copies of a segment of DNA
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DNA Replication vs. PCR
• PCR is a laboratory version of DNA Replication in cells
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• DNA replication is the copying of DNA
• It typically takes a cell just a few hours to copy all of its DNA
• DNA replication is semi-conservative (i.e. one strand of the
DNA is used as the template for the growth of a new DNA
strand)
• This process occurs with very few errors (on average there is
one error per 1 billion nucleotides copied)
• More than a dozen enzymes and proteins participate in DNA
replication …..DNA Polymerase, DNA Ligase, Primase,
Helicase, Topoisomerase, Single strand binding protein
Molecular Biology
• The laboratory version is commonly called “in vitro” since it occurs
in a test tube while “in vivo” signifies occurring in a living cell.
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1. Strands of template DNA (or RNA) are separated by
melting
2. Forward Primer binds to one strand of template, Reverse
Primer to other strand
3. DNA polymerase extends 3’ end of each primer, copying
template
4. Strands are separated by raising temperature, allowing
both original DNA and copies to act as templates
5. Repeat steps 2-4 many times
Molecular Biology
Basic Principles of PCR
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The three main steps of PCR
Step 2: Primers Anneal
At 40C- 65C, the primers anneal (or bind to) their complementary
sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain
At 72C, DNA Polymerase extends the DNA chain by adding nucleotides
to the 3’ ends of the primers.
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Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two strands are separated)
Molecular Biology
• The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.
• In a PCR reaction, the following series of steps is repeated 20-40 times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA
fragment of interest 100,000 fold)
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Molecular Biology
37-65°C
70-75°C
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94°C
Molecular Biology
The first round of PCR
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Molecular Biology
PCR increases the yield of DNA exponentially
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Molecular Biology
Exponential Amplification
30 cycles --- 1 billion copies in theory
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Components of PCR Reaction
Thermocyler
Template DNA
Flanking Primers
dNTP (dATP, dTTP, dCTP, dGTP)
Molecular Biology
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• building blocks for new DNA strands
• PCR Buffer (mg++)
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• PCR buffer provides optimal pH and
salt condition
• Thermo-stable polymerase
• Taq Polymerase
o DNA polymerase needs Mg++ as cofactor
o Each DNA polymerase works best under optimal
temperature, pH and salt concentration
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Thermus aquaticus
PCR Buffer
• Basic Components (Stabilizes the DNA polymerase, DNA, and nucleotides)
• Magnesium – Since Mg ions form complexes with dNTPs, primers and
DNA templates, the optimal concentration of MgCl2 has to be selected for
each experiment. Too few Mg2+ ions result in a low yield of PCR product,
and too many increase the yield of non-specific products and promote
misincorporation.
• Potential Additives
Molecular Biology
• 20mM Tris-HCL pH 8.4
• 50mM KCl
• 1.5 mM MgCl2
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dimethyl sulphoxide (DMSO),
dimethyl formamide (DMF),
urea
formamide
• Long Targets >1kb. Formamide and glycerol
• Low concentration of template: Polyethylene glycol (PEG)
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• Helix Destabilisers - useful when target DNA is high G/C With NAs of high
(G+C) content.
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Why Are Primers Important?
• Primers are what gives PCR its SPECIFICITY!!!
• e.g. hairpins, homodimers, heterodimers
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• Paired flanking primers
• Length (18-30bp) for general applications (30-35 bp for multiplex
PCR)
• GC content 50-60%
• GC clamp
• Tm’s between 50-60°C
• Avoid simple sequences – e.g. strings of G’s
• Avoid primer self complementary
Molecular Biology
• Good primer design: PCR works great.
• Bad primer design: PCR works terrible.
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Factor 1:
Melting / Annealing Temperature
• Longer primers stick better = melt at a
higher temperature.
• GC CONTENT
• More G-C content = more triple bonds =
primers stick better = melt at higher
temperature.
• PCR Annealing Temp = Tm - 20°C
Molecular Biology
• PRIMER LENGTH
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aagtcagtcagtactagtgatgta
aagtcagtcag
• Tm = [4(G + C) + 2(A + T)] °C
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• Forward Primer (EcoR I ) T(a)= 56
5- ATT GGA ATT C AC ATC CGA CAC AAA TGT TG -3
• Reverse Primer (Hind III) T(a)= 56
5- AAA GGT TAA GCT TTA ATT AGT TCT CTT CGG A -3
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4381 gcggcgcgaa tgaacatctt attggctatc acatccgaca caaatgttgc catcccattg
4441 cttaatcgaa taaaaatcag gctacatggg tgctaaatct ttaacgataa cgccattgag
4501 gctggtcatg gcgctcataa atctggtata cttaccttta cacattgggg ctgattctgg
4561 attcgacggg atttgcgaaa cccaaggtgc atgccga ggg gcggttggcc tcgtaaaaag
4621 ccgcaaaaaa tagtcgcaaa cgacgaaaac tacgctttag cagcttaata acctgcttag
4681 agccctctct ccctagcctc cgctcttagg acggggatca agagaggtca aacccaaaag
4741 agatcgcgtg gaagccctgc ctggggttga agcgttaaaa cttaatcagg ctagtttgtt
4801 agtggcgtgt ccgtccgcag ctggcaagcg aatgtaaaga ctgactaagc atgtagtacc
4861 gaggatgtag gaatttcgga cgcgggttca actcccgcca gctccaccaa aattctccat
4921 cggtgattac cagagtcatc cgatgaagtc ctaagagccc gcacggcgca agccctgcgg
4981 gcttttttgt gccctcaatt tgtcccgcga agtccgaaga gaactaatta aatccgaacc
5041 ttttaggccc attgataggc ccaacgaaaa gctctattgt ttacgttggg cctaaacgca
Molecular Biology
A gene sequence of E. coli MG1655 strain from (NCBI)
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Factor 2:
Complementarity
• Excessive similarity between
primers, especially at the 3’ ends,
leads to the formation of “primer
dimers”
• PRIMER-TARGET (GOOD)
atcggactatcga
tagcctgatagctatacttatggcca
• Ideally should be 100% similar for
maximal specificity.
• Primers don’t HAVE to be perfectly
similar to target to work.
Molecular Biology
gctatacttatggcca
• PRIMER-PRIMER (BAD)
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atcggactatcga
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Some related terms
Polymerases: enzymes that link individual nucleotides together to form
long DNA or RNA chains
Hybridisation (annealing:) the joining of two complementary DNA (or RNA)
strands to form a double strand
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Primer: a short DNA fragment with a defined sequence that serves as an
extension point for polymerases
Molecular Biology
Sequence: the order of the nucleotides in DNA (DNA sequence) or RNA
(RNA sequence)
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Quick
Reliable
Sensitive
Relatively easy
Specific
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Need for equipment
Taq polymerase is expensive
Contamination
False reactions
Internal control
Cross-reaction
Enrichment steps in
(contaminated) samples
• Capacity building needed
• Unspecific amplification
Molecular Biology
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Disadvantage of PCR
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Advantages of PCR
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Detection of specific genome
Classical PCR - with a primer pair
Colony PCR
Nested PCR - amplification of larger area then specific detection in
multiplied genome part (more sensitive)
Multiplex PCR- Amplifying various genes simultaneously (uses more
than one set of primers)
Real time PCR - uses fluorescent markers to track the number of
copies of target sequence produced in each cycle (to quantify the
amount of genome in sample)
Reverse Transcriptase PCR- Detection of RNA with reverse
transcriptase enzyme
Screening specific genes for unknown mutations
Genotyping using short primers or primer pairs that are often
repeated in the genome
Molecular Biology
Applications of PCR
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Uses of PCR
• Forensics:
 PCR’s ability to amplify even the smallest amount of DNA from samples collected
at a crime scene gives the method great power when used in criminal forensics.
• Paternity Testing
 By amplifying specific DNA fragments from parents or close relatives, it is
possible to reconstruct relatedness between individuals. also PCR used to
identify historical family relationships!
• Archaeology
 Reconstructing the Dead Sea Scrolls.
 Identification of paint pigments in cave paintings.
 Determining relatedness between individuals in ancient ossuaries.
 Constructing dinosaurs from blood preserved in amber specimens. (!)
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 PCR can be used to accurately measure the exact quantity of geneticallymodified food in a shipment, by “looking” at the DNA that makes up the
food!
Molecular Biology
• GMO Food Detection
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Uses of PCR
• Disease Diagnosis
 real-time PCR is used to directly monitor the amount of HIV virus in patients
suffering from infection. By monitoring the amount of virus present, the
drug therapy can be continually adjusted to maximize virus suppression.
• Basic Research
 Quantitative PCR is a key component of determining the levels of gene
expression, and is a critical tool in cancer research, disease studies, and
developmental biology.
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• Disease Treatment
Molecular Biology
 PCR can identify disease-causing organisms much earlier than other
methods, since it looks for the DNA of the organism itself, not its proteins or
its effect on our immune system.
 PCR has even been used to diagnose diseases of the past, by amplifying
minute amounts of disease-related DNA in preserved specimens.
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• Can You amplify RNA by PCR?
• Do PCR reaction needs a source of energy?
• What is the difference between amplification of genome
invitro and invivo?
Molecular Biology
Questions?
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Restriction Digestion Result
Molecular Biology
Group B
Group C
EcoRI HindIII SmaI EcoRI HindIII SmaI
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1500bp
Group A
Ladder plasmid EcoRI HindIII Sma I
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