PCR
Mr. S.Ghosh
MITS
Division of Biotechnology
Laboratory Tools and Techniques
The methods used by molecular biologists to
study DNA have been developed through
adaptation of the chemical reactions and
biological processes that occur naturally in cells
Many of the enzymes that copy DNA, make
RNA from DNA, and synthesize proteins from
an RNA template were first characterized in
bacteria. This basic research has become
fundamental to our understanding of the
function of cells and have led to immense
practical applications for studying a gene and
its corresponding protein.
As science advances, so do the number of tools
available that are applicable to the study of
molecular genetics.
PCR
The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying
relatively large quantities of DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that
is used by the bacterium Thermus aquaticus that was discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
PCR
The cycling reactions :
There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is
done on an automated Thermo Cycler, which can heat and cool the reaction tubes in
a very short time.
Denaturation at around 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all
enzymatic reactions stop (for example the extension from a previous cycle).
Annealing at around 54°C :
Hydrogen bonds are constantly formed and broken between the single stranded primer
and the single stranded template. If the primers exactly fit the template, the hydrogen
bonds are so strong that the primer stays attached
Extension at around 72°C :
The bases (complementary to the template) are coupled to the primer on the 3' side
(the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,
bases are added complementary to the template)
PCR
The different steps of PCR
PCR
Exponential increase of the number of copies during PCR
PCR
Every cycle results in a doubling of the number of
strands DNA present
After the first few cycles, most of the product DNA
strands made are the same length as the distance
between the primers
The result is a dramatic amplification of a the DNA
that exists between the primers. The amount of
amplification is 2 raised to the n power; n represents
the number of cycles that are performed. After 20
cycles, this would give approximately 1 million fold
amplification. After 40 cycles the amplification would
be 1 x 1012
PCR and Contamination
The most important consideration in PCR is contamination
Even the smallest contamination with DNA could affect
amplification
For example, if a technician in a crime lab set up a test
reaction (with blood from the crime scene) after setting up a
positive control reaction (with blood from the suspect) cross
contamination between the samples could result in an
erroneous incrimination, even if the technician changed
pipette tips between samples. A few blood cells could
volitilize in the pipette, stick to the plastic of the pipette,
and then get ejected into the test sample
Modern labs take account of this fact and devote
tremendous effort to avoiding cross-contamination
Optimizing PCR protocols
PCR can be very tricky
While PCR is a very powerful technique,
often enough it is not possible to achieve
optimum results without optimizing the
protocol
Critical PCR parameters:
- Concentration of DNA template,
nucleotides, divalent cations
(especially Mg2+) and polymerase
- Error rate of the polymerase (Taq, Vent
exo, Pfu)
- Primer design
Primer design
General notes on primer design in PCR
Perhaps the most critical parameter for successful PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length
- specificity and the temperature of annealing are at
least partly dependent on primer length
- oligonucleotides between 20 and 30 (50) bases are highly sequence specific
- primer length is proportional to annealing efficiency: in general, the longer the
primer, the more inefficient the annealing
- the primers should not be too short as specificity decreases
Primer design
Specificity
Primer specificity is at least partly dependent on primer length: there are many more
unique 24 base oligos than there are 15 base pair oligos
Probability that a sequence of length n will occur randomly in a sequence of length m
is:
Example: the mtDNA genome has about 20,000 bases, the probability
of randomly finding sequences of length n is:
P = (m – n +1) x (¼)n
n
5
10
15
Pn
19.52
1.91 x 10-2
1.86 x 10-5
Primer design
Complementary primer sequences
- primers need to be designed with absolutely no intra-primer homology beyond 3 base
pairs. If a primer has such a region of self-homology, “snap back” can occur
- another related danger is inter-primer homology: partial homology in the middle
regions of two primers can interfere with hybridization. If the homology should occur
at the 3' end of either primer, primer dimer formation will occur
G/C content
- ideally a primer should have a near random mix of nucleotides, a 50% GC content
- there should be no PolyG or PolyC stretches that can promote non-specific annealing
3’-end sequence
- the 3' terminal position in PCR primers is essential for the control of mis-priming
- inclusion of a G or C residue at the 3' end of primers helps to ensure correct binding
(stronger hydrogen bonding of G/C residues)
Primer design
Melting temperature (Tm)
- the goal should be to design a primer with an annealing temperature of at least 50°C
- the relationship between annealing temperature and melting
temperature is one of the “Black Boxes” of PCR
- a general rule-of-thumb is to use an annealing temperature that is 5°C lower
than the melting temperature
- the melting temperatures of oligos are most accurately calculated using nearest
neighbor thermodynamic calculations with the formula:
Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log 10 [K+]
(H is the enthalpy, S is the entropy for helix formation, R is the molar gas
constant and c is the concentration of primer)
- a good working approximation of this value can be calculated using the Wallace formula:
Tm = 4x (#C+#G) + 2x (#A+#T) °C
- both of the primers should be designed such that they have similar melting temperatures.
If primers are mismatched in terms of Tm, amplification will be less efficient or may not
work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with
the lower Tm may not work at higher temperatures.
The PCR Process—Reaction
Components
Typical components of a PCR include:
◦ DNA: the template used to synthesize new DNA
strands.
◦ DNA polymerase: an enzyme that synthesizes new
DNA strands.
◦ Two PCR primers: short DNA molecules
(oligonucleotides) that define the DNA sequence to
be amplified.
◦ Deoxynucleotide triphosphates (dNTPs): the
building blocks for the newly synthesized DNA
strands.
◦ Reaction buffer: a chemical solution that provides
the optimal environmental conditions.
◦ Magnesium: a necessary cofactor for DNA
polymerase activity.
HPCR Amplify DNA?

One PCR cycle consists of a DNA denaturation
step, a primer annealing step and a primer
extension step.
DNA Denaturation: Expose the DNA template to high
temperatures to separate the two DNA strands and allow
access by DNA polymerase and PCR primers.
Primer Annealing: Lower the temperature to allow
primers to anneal to their complementary sequence.
Primer E xtension: Adjust the temperature for optimal
thermostable DNA polymerase activity to extend primers.

PCR uses a thermostable DNA polymerase so
that the DNA polymerase is not heat-inactivated
during the DNA denaturation step. Taq DNA
polymerase is the most commonly used DNA
polymerase for PCR.
◦ DNA polymerase extends the primer by
sequentially adding a single dNTP (dATP, dGTP,
dCTP or dTTP) that is complementary to the
existing DNA strand
◦ The sequence of the newly synthesized strand
is complementary to that of the template
strand.
◦ The dNTP is added to the 3´ end of the
growing DNA strand, so DNA synthesis occurs
in the 5´ to 3´ direction.
Mechanism of DNA Synthesis
Instrumentation

Thermal cyclers have a heat-conducting
block to modulate reaction temperature.
◦ Thermal cyclers are programmed to maintain
the appropriate temperature for the required
length of time for each step of the PCR cycle.
◦ Reaction tubes are placed inside the thermal
cycler, which heats and cools the heat block to
achieve the necessary temperature.
A typical thermal cycler program is:
Initial DNA denaturation at 95°C for 2 minutes
20–35 PCR cycles:
Denaturation at 95°C for 30 seconds to 1 minute
Annealing at 42–65°C for 1 minute
Extension at 68–74°C for 1–2 minutes
Final extension at 68–74°C for 5–10 minutes
Soak at 4°C
Thermal Cycling Programs
Many PCR parameters might need to be
optimized to increase yield, sensitivity of
detection or amplification specificity.
These parameters include:
 Magnesium concentration
 Primer annealing temperature
 PCR primer design
 DNA quality
 DNA quantity
PCR Optimization


Magnesium concentration is often one of the
most important factors to optimize when
performing PCR.
The optimal Mg2+ concentration will depend upon
the primers, template, DNA polymerase, dNTP
concentration and other factors.
◦ Some reactions amplify equally well at a number of Mg2+
concentrations, but some reactions only amplify well at a
very specific Mg2+ concentration.

When using a set of PCR primers for the first
time, titrate magnesium in 0.5 or 1.0mM
increments to empirically determine the optimal
Mg2+ concentration.
Magnesium Concentration



PCR primers must anneal to the DNA template at
the chosen annealing temperature.
The optimal annealing temperature depends on
the length and nucleotide composition of the PCR
primers
The optimal annealing temperature is often
within 5°C of the melting temperature (Tm) of the
PCR primer
The melting temperature is defined as the temperature at
which
50% of complementary DNA molecules will be annealed
(i.e., double-stranded).

When performing multiplex PCR, where multiple
DNA targets are amplified in a single PCR, all sets
of PCR primers must have similar annealing
temperatures.
Primer Annealing Temperature
DNA Quality

DNA should be intact and free of
contaminants that inhibit amplification.
◦ Contaminants can be purified from the original
DNA source.
 Heme from blood, humic acid from soil and
melanin from hair
◦ Contaminants can be introduced during the
purification process.
 Phenol, ethanol, sodium dodecyl sulfate (SDS)
and other detergents, and salts.
DNA quantity
 More template is not necessarily better.
◦ Too much template can cause nonspecific
amplification.
◦ Too little template will result in little or no PCR
product.

The optimal amount of template will
depend on the size of the DNA molecule.
DNA Quantity
PCR and RT-PCR have hundreds of
applications. In addition to targeting and
amplifying a specific DNA or RNA sequence,
some common uses include:
 Labeling DNA or RNA molecules with tags,
such as fluorophores or radioactive labels, for
use as tools in other experiments.
 Cloning a DNA or RNA sequence
 Detecting DNA and RNA
 Quantifying DNA and RNA
 Genotyping and DNA-based identification
Applications of PCR

Labeling DNA with tags for use as tools
(probes) to visualize complementary DNA or
RNA molecules.
◦ Radioactive labels.
 Radioactively labeled probes will darken an X-ray film.
◦ Fluorescent labels (nonradioactive)
 Fluors will absorb light energy of a specific wavelength
(the excitation wavelength) and emit light at a
different wavelength (emission wavelength).
 The emitted light is detected by specialized
instruments such as fluorometers.
Labeling DNA
Quantitative PCR

Avoids problems associated with the plateau
effect, which reduces amplification efficiency
and limits the amount of PCR product
generated due to depletion of reactants,
inactivation of DNA polymerase and
accumulation of reaction products.
◦ The result of the plateau effect is that the amount
of PCR product generated is no longer proportional
to the amount of DNA starting material.
◦ The plateau effect becomes more pronounced at
higher cycle numbers.

Often performed in real time to monitor the
accumulation of PCR product at each cycle.
◦ Real-time PCR allows scientists to quantify DNA
before the plateau effect begins to limit PCR product
synthesis.
RT PCR
THE PROBLEM
• NEED TO QUANTITATE DIFFERENCES
IN mRNA EXPRESSION
• SMALL AMOUNTS OF mRNA
– LASER CAPTURE
– SMALL AMOUNTS OF TISSUE
– PRIMARY CELLS
– PRECIOUS REAGENTS
28
THE PROBLEM
• QUANTITATION OF mRNA
–
–
–
–
northern blotting
ribonuclease protection assay
in situ hybridization
PCR
•
•
•
•
most sensitive
can discriminate closely related mRNAs
technically simple
but difficult to get truly quantitative results using
conventional PCR
29
NORTHERN
control
expt
target gene
internal control gene
actin, GAPDH, RPLP0 etc
Corrected fold increase = 10/2 = 5
Ratio target gene in experimental/control = fold change in target gene
fold change in reference gene
30
same copy number in all cells
 expressed in all cells
 medium copy number advantageous

◦ correction more accurate
Standards
31
AMOUNT OF DNA
1600000000
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
1400000000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
AMOUNT OF DNA
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
5
10
15
20
25
PCR CYCLE NUMBER
30
35
32
AMOUNT OF DNA
1600000000
1400000000
1200000000
1000000000
800000000
600000000
400000000
AMOUNT OF DNA
1600000000
200000000
0
1400000000
0
1200000000
5
10
20
15
25
30
35
PCR CYCLE NUMBER
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
33
AMOUNT OF DNA
AMOUNT OF DNA
10000000000 10000000000
1000000000 1000000000
100000000 100000000
10000000
10000000
1000000
1000000
100000
100000
10000
10000
1000
1000
100
100
10
10
1
1
0
5
010
515
1020
1525
2030
2535
30
35
PCR CYCLE NUMBER
PCR CYCLE NUMBER
34
Linear ~20 to ~1500
35
Linear ~20 to ~1500
36
REAL TIME PCR
• kinetic approach
• early stages
• while still linear
37
www.biorad.com
3.
intensifie
r
1. halogen
tungsten
lamp
2b. emission
filters
2a.
excitation
filters
5. ccd
detect
or
350,00
0
pixels
4. sample
plate
38
SERIES OF 10-FOLD DILUTIONS
39
40
41
SERIES OF 10-FOLD DILUTIONS
42
threshold
Ct
SERIES OF 10-FOLD DILUTIONS
43
threshold = 300
44
threshold
SERIES OF 10-FOLD DILUTIONS
15
45
STANDARD CURVE
METHOD
46
PFAFFL METHOD
– M.W. Pfaffl, Nucleic Acids
Research 2001 29:2002-2007
47
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
8
7
6
5
4
16
13
10
8
5
32
25
19
14
6
64
47
34
24
7
128
89
61
41
8
256
170
110
70
9
512
323
198
119
10
1,024
613
357
202
11
2,048
1,165
643
343
12
4,096
2,213
1,157
583
13
8,192
4,205
2,082
990
14
16,384
7,990
3,748
1,684
15
32,768
15,181
6,747
2,862
16
65,536
28,844
12,144
4,866
17
131,072
54,804
21,859
8,272
18
262,144
104,127
39,346
14,063
19
524,288
197,842
70,824
23,907
20
1,048,576
375,900
127,482
40,642
21
2,097,152
714,209
229,468
69,092
22
4,194,304
1,356,998
413,043
117,456
23
8,388,608
2,578,296
743,477
199,676
24
16,777,216
4,898,763
1,338,259
339,449
25
33,554,432
9,307,650
2,408,866
577,063
26
67,108,864
17,684,534
4,335,959
981,007
27
134,217,728
33,600,615
7,804,726
1,667,711
28
268,435,456
63,841,168
14,048,506
2,835,109
29
536,870,912
121,298,220
25,287,311
4,819,686
30
1,073,741,824
230,466,618
45,517,160
8,193,466
1,200,000,000
1,000,000,000
800,000,000
AFTER 1 CYCLE
100%
= 2.00x
600,000,000
90% = 1.90x
400,000,000
80%
= 1.80x
70%
= 1.70x
200,000,000
0
0
10
48
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
8
7
6
5
4
16
13
10
8
5
32
25
19
14
6
64
47
34
24
7
128
89
61
41
8
256
170
110
70
9
512
323
198
119
10
1,024
613
357
202
11
2,048
1,165
643
343
12
4,096
2,213
1,157
583
13
8,192
4,205
2,082
990
14
16,384
7,990
3,748
1,684
15
32,768
15,181
6,747
2,862
16
65,536
28,844
12,144
4,866
17
131,072
54,804
21,859
8,272
18
262,144
104,127
39,346
14,063
19
524,288
197,842
70,824
23,907
20
1,048,576
375,900
127,482
40,642
21
2,097,152
714,209
229,468
69,092
22
4,194,304
1,356,998
413,043
117,456
23
8,388,608
2,578,296
743,477
199,676
24
16,777,216
4,898,763
1,338,259
339,449
25
33,554,432
9,307,650
2,408,866
577,063
26
67,108,864
17,684,534
4,335,959
981,007
27
134,217,728
33,600,615
7,804,726
1,667,711
28
268,435,456
63,841,168
14,048,506
2,835,109
29
536,870,912
121,298,220
25,287,311
4,819,686
30
1,073,741,824
230,466,618
45,517,160
8,193,466
1,200,000,000
1,000,000,000
800,000,000
AFTER 1 CYCLE
100%
= 2.00x
600,000,000
90% = 1.90x
400,000,000
80%
= 1.80x
70%
= 1.70x
200,000,000
0
0
10
AFTER N CYCLES:
fold increase =
(efficiency)n
49
1,200,000,000
1,200,000,000
100% EFF
90% EFF
80% EFF
70% EFF
1,000,000,000
AMOUNT OF DNA
1,000,000,000
800,000,000
800,000,000
600,000,000
400,000,000
600,000,000
200,000,000
400,000,000
0
0
200,000,000
10
20
30
0
0
10
20
30
PCR CYCLE NUMBER
10,000,000,000
100% EFF
90% EFF
80% EFF
70% EFF
1,000,000,000
AMOUNT OF DNA
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
8
7
6
5
4
16
13
10
8
5
32
25
19
14
6
64
47
34
24
7
128
89
61
41
8
256
170
110
70
9
512
323
198
119
10
1,024
613
357
202
11
2,048
1,165
643
343
12
4,096
2,213
1,157
583
13
8,192
4,205
2,082
990
14
16,384
7,990
3,748
1,684
15
32,768
15,181
6,747
2,862
16
65,536
28,844
12,144
4,866
17
131,072
54,804
21,859
8,272
18
262,144
104,127
39,346
14,063
19
524,288
197,842
70,824
23,907
20
1,048,576
375,900
127,482
40,642
21
2,097,152
714,209
229,468
69,092
22
4,194,304
1,356,998
413,043
117,456
23
8,388,608
2,578,296
743,477
199,676
24
16,777,216
4,898,763
1,338,259
339,449
25
33,554,432
9,307,650
2,408,866
577,063
26
67,108,864
17,684,534
4,335,959
981,007
27
134,217,728
33,600,615
7,804,726
1,667,711
28
268,435,456
63,841,168
14,048,506
2,835,109
29
536,870,912
121,298,220
25,287,311
4,819,686
30
1,073,741,824
230,466,618
45,517,160
8,193,466
100,000,000
10,000,000
1,000,000
100,000
10,000
1,000
100
10
1
0
10
20
PCR CYCLE NUMBER
50
30
10,000,000,000
100% EFF
90% EFF
80% EFF
70% EFF
AMOUNT OF DNA
1,000,000,000
100,000,000
10,000,000
1,000,000
100,000
10,000
1,000
100
10
1
0
10
20
PCR CYCLE NUMBER
30
51
SERIES OF 10-FOLD DILUTIONS
52
threshold
SERIES OF 10-FOLD DILUTIONS
15
53
QUALITY CONTROL EFFICIENCY CURVES








use pcr baseline subtraction (not curve fitting
default option) - see next slide
set the threshold manually to lab standard
check all melting curves are OK
check slopes are parallel in log view
delete samples if multiple dilutions cross line
together (usually at dilute end of curve)
delete samples if can detect amplification at
cycle 10 or earlier
make sure there are 5 or more points
check correlation coefficient is more than
1.990
54
55
Quality Control








use pcr baseline subtraction (not curve fitting
default option)
set the threshold manually to lab standard
check all melting curves are OK
check slopes are parallel in log view
delete samples if multiple dilutions cross line
together (usually at dilute end of curve)
delete samples if can detect amplification at
cycle 10 or earlier
make sure there are 5 or more points
check correlation coefficient is more than
1.990
56
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
57
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
58
Should be free of protein (absorbance
260nm/280nm)
 Should be undegraded (28S/18S ~2:1)
 Should be free of DNA (DNAse treat)
 Should be free of PCR inhibitors

◦ Purification methods
◦ Clean-up methods
59
OVERVIEW
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
60

Oligo (dt)

Random hexamer (NNNNNN)

Specific
Importance of reverse
transcriptase primers
61

adds a bias to the results

efficiency usually not known
Reverse Transcription
62
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
63
specific
 high efficiency
 no primer-dimers
 Ideally should not give a DNA signal

◦ cross exon/exon boundary
Importance of primers in PCR
64
Number Games
Nested PCR
Hot Start PCR
Multiplex PCR
RT PCR
THANK
U