Polymerase Chain Reaction

Darius Noel C. Minoza

College of Agriculture
Western Mindanao State University
Lecture Outline
History of PCR
Underlying technology of PCR
Major application of PCR
Southern Blotting
Western Blotting
Northern Blotting
Learning Outcomes
Understand the technology that drives PCR
and real-time PCR
Be aware of common hybridization techniques
and understand how they differ
The Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) revolutionized life
sciences as it provides a sensitive, reliable, efficient, and
convenient means of amplifying relatively large quantities of
DNA
Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993
The technique was made possible by the discovery of Taq
polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus, discovered in hot springs.
The primary materials used in PCR:
- DNA nucleotides: the building blocks for the new DNA
- Template DNA: the DNA sequence that you want to amplify
- Primers: single-stranded short DNA (16--50 nucleotides
long) that are complementary to a short region on either end of
the template DNA
- DNA polymerase: a heat stable enzyme that catalyzes the
synthesis of new DNA
PCR: the technology that changed the world we knew
Primers dictate the successfulness of a PCR
Specificity?
Proper
annealing to
the template?
Tricks of the Trade
Thermophillus aquaticus Taq polymerase
The Polymerase Chain Reaction
Beginning with a single molecule of the genetic
material DNA, the PCR can generate 100
billion similar molecules in an afternoon. The
reaction is easy to execute. It requires no
more than a test tube, a few simple reagents,
and a source of heat. Karry Mullis, 1990
Scientific American

PCR
PCR is, in essence, marvellously simple.
The underlying mechanism of PCR is based upon
our understanding of nucleic acid biochemistry.
Karry Mullis realized that to make DNA you need
4 things:
A template
Some primers
A DNA polymerase
Deoxynucleotides
Purine
Py r i m i di ne
Guanine Adenine
Cytosine Thymine Uracil
Molecular techniques are based on the structure of DNA and RNA
Thymine Adeni ne
Cyt osine
Guanine
Adenine
Guanine
Thymine
Cytosine
Need:
Target DNA
Primers: 17 to 30bp, GC content >50%
Primers can be for universal conserved sequences (16S rDNA,
dehydrogenase genes) or genus-level conserved sequences (Nod, Rhl, LamB
genes)
dNTPs
DNA polymerase (original was taq polymerase from Thermus aquaticus.
Now there are several other DNA polymerases available)
PCR-Polymerase Chain Reaction

In many cases there is not enough DNA in a sample for a gene probe to detect.
Sample DNA can be amplified using PCR.
Primer annealing
5'
5'
3'
3'
target DNA
repeat PCR cycles
5'
5'
3'
3'
Double-stranded DNA
Denaturation
5'
5'
3'
3'
Extension 3'
5'
5'
5'
5' 3'
3'
3'
3'
5'
5'
5'
5' 3'
3'
3'
Extension
PCR Round 1
DNA polymerase always adds nucleotides to
the 3 end of the primer
denaturation
primer annealing
extension
PCR Round 2
Chromosomal strand
Long strand
After the second round of
PCR, the number of long
strands increases
arithmetically and the
number of short strands
increases exponentially (the
number of chromosomal
strands is always the same).
5'
5'
3'
3'
5'
5'
3'
3'
3'
5'
5'
5'
5'
3'
3'
3'
3'
5'
5'
5'
5' 3'
3'
3'
3' 5'
3'
5'
5' 3'
5'
5' 3'
3'
3'
5'
5'
3'
Short strand
72
0
C - primer extension
94
0
C - denaturation
Temperature
0
C
Temperature control in a PCR thermocycler

94
0
C - denaturation
50 70
0
C - primer annealing

# PCR cycles
After 25 cycles have 3.4 x 10
7
times more DNA

plateau is reached after
25-30 cycles

Differences between DNA
Replication & PCR
No Helicase or Topoisomerase PCR uses
the first heat step to completely separate
the strands of DNA
No Primase primers are already made
DNA primers (not RNA) no need for DNA
Polymerase I
No leading or lagging strands DNA is
completely unzipped, no Okazaki fragments
How does a PCR work?
Denature template (94C)
Anneal primers (specific to primer sets but
normally approximately 55C)
Extension (72C for Taq polymerase)
Turn 1 DNA sequence into 1,000,000,000 in
just 30 cycles
The Reagents
Reaction buffer
Magnesium chloride
DNA template
Oligonucleotide primers
Taq polymerase
dNTPs
Water
Reaction Buffer
Reaction buffer RATIONALE
Stabilizes the DNA
polymerase
Supplied as a 10x buffer
that generally consists of
500 mM KCl and 100 mM
Tris-HCl
Magnesium Chloride
MgCl Rationale
Mg
2+
is an essential co-
factor of DNA polymerase.
Too little of this is in the
reaction impairs DNA
polymerase activity,
however too much can
result in non-specific
priming during the PCR
Generally, included at a final
concentration of 1-3 mM
Template DNA
Template Rationale
The template must be DNA
Also, the template must
contain the region to be
amplified
Miniscule amount of impure
sample will suffice,
however, problems arise if
sample contains impurities
that may inhibit DNA
polymerase
Oligonucleotide primers
Primers Rationale
Primers should be 100%
specific for the region to be
amplified. A forward and
reverse primer is needed
The reverse primer is
designed to be
complementary and the
reverse of the coding
strand.
Primers should have similar
annealing temperatures
Taq polymerase
Taq Rationale
The enzyme responsible for
synthesizing the amplified
DNA
Usually Taq polymerase,
although other heat-stable
polymerases are used
dNTPs
dNTPs Rationale
The dNTPs are added to the
growing chain by DNA
polymerase
They are activated
dinucleotide triphosphates
(dATP, dGTP, dCTP, and
dTTP)
Generaly used at a final
concentration of 200 uM
Water
Water Rationale
Reaction medium. Carrying
out PCR reactions in larger
volumes can help with
impurities
Primer Design
A specific forward and reverse primer are
required
The reverse primer must prime 35 and
therefore is the reverse and complimentary
sequence of the target
Primers should have comparable annealing
temperatures
Many algorithms are available online to aid
both in primer design and analysis
Before you design your own primers
Dont reinvent the wheels!
Before you start designing primers
Find and use the right resources!
What are the primers for?
General purpose amplification?
SNPs detection/validation?
Methylation study?
Real-time PCR?
Microarray probes?
Degenerate PCR?
Multiplex PCR?
What do you have to begin with?
Single DNA/protein sequence?
Multiple DNA/protein sequence files?
GenBank ID/Gene ID/Gene Symbol/rsSNP ID?
After you have your primers designed
Consider a second opinion!
Most likely your primers can be designed by
several different software
Different software may vary significantly in:
Concepts and overall approaches
Designing criteria and default settings
Comprehensiveness
Usability
Accessibility and speed

Consider a second opinion when
You are new to such design task/application
You dont have a lot of confidence in the initial result
General rules for primer design
-- Primer and amplicon length
Primer length determines the specificity and
significantly affect its annealing to the template
Too short -- low specificity, resulting in non-specific
amplification
Too long -- decrease the template-binding efficiency at
normal annealing temperature due to the higher probability
of forming secondary structures such as hairpins.
Optimal primer length
18-24 bp for general applications
30-35 bp for multiplex PCR
Optimal amplicon size
300-1000 bp for general application, avoid > 3 kb
50-150 bp for real-time PCR, avoid > 400 bp
General rules for primer design
-- Melting temperature (T
m
)
T
m
is the temperature at which 50% of the DNA duplex
dissociates to become single stranded
Determined by primer length, base composition and concentration.
Also affected by the salt concentration of the PCR reaction mix
Working approximation: T
m
=2(A+T)+4(G+C) (suitable only for 18mer
or shorter).
Optimal melting temperature
52C-- 60C
T
m
above 65C should be generally avoided because of the potential for
secondary annealing.
Higher T
m
(75C-- 80C) is recommended for amplifying high GC
content targets.
Primer pair T
m
mismatch
Significant primer pair T
m
mismatch can lead to poor amplification
Desirable T
m
difference < 5C between the primer pair
General rules for primer design
-- Specificity and cross homology
Specificity
Determined primarily by primer length as well as sequence
The adequacy of primer specificity is dependent on the nature of the
template used in the PCR reaction.
Cross homology
Cross homology may become a problem when PCR template is genomic
DNA or consists of mixed gene fragments.
Primers containing highly repetitive sequence are prone to generate non-
specific amplicons when amplifying genomic DNA.
Avoid non-specific amplification
BLASTing PCR primers against NCBI non-redundant sequence database
is a common way to avoid designing primers that may amplify non-
targeted homologous regions.
Primers spanning intron-exon boundaries to avoid non-specific
amplification of gDNA due to cDNA contamination.
Primers spanning exon-exon boundaries to avoid non-specific
amplification cDNA due to gDNA contamination.
General rules for primer design
-- GC content; repeats and runs
Primer G/C content
Optimal G/C content: 45-55%
Common G/C content range: 40-60%

Runs (single base stretches)
Long runs increases mis-priming (non-specific annealing)
potential
The maximum acceptable number of runs is 4 bp

Repeats (consecutive di-nucleotide)
Repeats increases mis-priming potential
The maximum acceptable number of repeats is 4 di-
nucleotide
General rules for primer design
-- Primer secondary structures
Hairpins
Formed via intra-molecular interactions
Negatively affect primer-template binding, leading to poor or no
amplification
Acceptable G (free energy required to break the structure): >-2
kcal/mol for 3end hairpin; >-3 kcal/mol for internal hairpin;
Self-Dimer (homodimer)
Formed by inter-molecular interactions between the two same primers
Acceptable G: >-5 kcal/mol for 3end self-dimer; >-6 kcal/mol for
internal self-dimer;
Cross-Dimer (heterodimer)
Formed by inter-molecular interactions between the sense and antisense
primers
Acceptable G: >-5 kcal/mol for 3end cross-dimer; >-6 kcal/mol for
internal cross-dimer;
General rules for primer design
-- Annealing temperatures and other considerations
T
a
(Annealing temperature) vs. T
m
T
a
is determined by the T
m
of both primers and amplicons:
optimal T
a
=0.3 x T
m
(primer)+0.7 x T
m
(product)-25
General rule: T
a
is 5C lower than T
m
Higher T
a
enhances specific amplification but may lower yields
Crucial in detecting polymorphisms
Primer location on template
Dictated by the purpose of the experiment
For detection purpose, section towards 3 end may be preferred.
When using composite primers
Initial calculations and considerations should emphasize on the template-
specific part of the primers
Consider nested PCR
http://www.hsls.pitt.edu/guides/genetics/obrc
http://www.usc.edu/hsc/nml/lib-services/bioinformatics/index.html
http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-meta?input-form=molbio-simple&query=pcr+primer&v%3Asources=OBRC&v%3Aproject=molbio
Resources for General Purpose PCR Primer Design
Primer3
Primer3Plus
PrimerZ
PerlPrimer
Vector NTI Advantage 10
General Purpose PCR Primer Design Tool Primer3
Web Site:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1043858198/info
Name Primer3 -- an online tool for PCR primer design
Type Web-based software
Key Functions Design PCR primers and hybridization probes.
Publication Info Methods Mol Biol 2000
Times Cited 823
Pros The original and most widely used PCR primer design program; uses sequence
as input; a huge number of options for customizing primer design;
Cons busy interface;
Note In OBRC; the program has been widely adopted by many primer design
software.
YiBus Rating 4 out of 5
General Purpose PCR Primer Design Tool Primer3Plus
Web Site:
http://www.bioinformatics.nl/primer3plus
More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1191263055/info

Name Primer3Plus, an enhanced web interface to Primer3
Type Web-based software
Key Functions Design PCR primers for a given DNA sequence.
Publication Info NAR 2007
Times Cited N/A
Pros Uses sequences or sequence file as input; a huge number of configuration
options; automates specific tasks such as designing primers for cloning or step-
wise sequencing; primers can be sent to an order form; clean, intuitive and well
organized interface;
Cons
Note in OBRC. It is an updated, task-oriented web-interface to the original Primer3.
YiBus Rating 4.5 out of 5
What is real-time PCR?
PCR or Polymerase Chain Reaction is a process
for the amplification of specific fragments of
DNA
Real-time PCR a specialized technique that
allows a PCR reaction to be visualized in real
time as the reaction progresses
Real-time PCR allows us to measure minute
amounts of DNA sequences in a sample
Conventional vs. Real-time
Conventional PCR tells us what
Real-time PCR tells us how much
Real-PCR has become a cornerstone of
molecular biology
Forensics
Sample identification and quantification
Animal and plant breeding
Gene copy number
Food testing
Percent GMO food
Disease diagnosis and management
Viral quantification
Gene expression analysis
Cancer and drug research
BRCA1 Expression Profiling
BRCA1 is a gene involved in tumor suppression
BRCA1 controls the expression of other genes
In order to monitor level of expression of
BRCA1, real-time PCR is used
HIV treatment
Drug treatment for HIV infection often
depends on monitoring the viral load
Rea;-time PCR allows direct measurement of
the amount of virus RNA in the patient
Determining percentage of GMO food
content
Determination of percent GMO food content
important for import/export regulations
Laboratories use real-time PCR to measure the
amount of transgenic versus wild-type DNA
Forensic Analysis
DNA quantification
Since standard forensic STR genotyping requires
defined amounts of DNA, real-time PCR can be
used to accurately quantify the amount of DNA in
an unknown sample
Stain identification
New real-time methods can be directly used to
identify the composition of unknown stains, with
much better accuracy than traditional color
change tests

What is in your tube at cycle 25?
A soup of nucleotides, primers, template,
amplicons (the amplified DNA product),
enzyme, etc.
1,000,000 copies of the amplicon right now
Thought experiments
What was it like last cycle, 24?
Almost exactly the same, except there were only
500,000 copies of the amplicon
And the cycle before that, 23?
Almost the same, but only 250,000 copies of the
amplicon
And what about cycle 22?
Not a whole lot different. 125,000 copies of the
amplicon
Thought Experiments
Whats it going to be like after the next cycle, in
cycle 26?
Probably there will be 2,000,000 amplicons
And cycle 27?
Maybe 4,000,000 amplicons
And at CYCLE 200?
In theory, there would be
100000000000000000000000000000000000000
0000000000000000000000000000000 amplicons
or 10^35 tons of DNA
Thought Experiments
To put this in perspective,
that would be equivalent to
the weight of ten billion
planets the size of Earth
Why not possible?
A clump of DNA the size of ten billion planets
wont quite fit in our PCR tube anymore!!!!
Realistically, as the chain reaction progresses,
it gets exponentially harder to find primers,
and nucleotides. And the polymerase is
wearing out.
So, exponential growth does not go on
forever!
How can all this be used to measure
DNA quantities?

Thought Experiments
Lets imagine that
you start with four
times as much DNA
as I do
Picture out two
tubes at cycle 25 and
work backwards a
few cycles
Cycle Me You
25 1,000,000 4,000,000
24 500,000 2,000,000
23 250,000 1,000,000
Thought Experiments
So if you started with
FOUR times as much
DNA as I did.....
Then youd reach
1,000,000 copies
exactly TWO cycles
earlier than I would!


Thought Experiments
What if YOU
started with
EIGHT times
less DNA
template than
I did?
Cycle Me You
25 1,000,000 125,000
26 2,000,000 250,000
27 4,000,000 500,000
28 8,000,000 1,000,000
Thought Experiments
What if YOU started
with EIGHT times LESS
DNA template than I
did?
Youd only have 125,000
copies right now at
cycle 25....
And youd reach
1,000,000 copies
exactly THREE cycles
later than I would!


We can easily see that
the left-right shift in the
curves is related to the
starting quantity of
DNA!
Ct (Cycle threshold)
values identify the
curve positions
DNA quant