Polymerase Chain Reaction

(PCR)

Dr Cath ONeill
PCR
A means of selectively amplifying a
particular segment of DNA in vitro. The
DNA to be amplified can be a whole gene
or part of a gene. Often the DNA to be
amplified is part of a large and complex
mixture.


Amplification is very specific and
sensitive!
5cgattagggttacgtagctagcaatgacgtatacacatgacga
tatataggtgaccagtacgatataagttgaccagtgatacacata
gatagtacacatgtatatagtttggaaccagacagatagacagg
tttggacagatgattaacccttaaggggacacactgtggggttac
atgatactagatacacagatacagataagggttacctagatct3


PCR can amplify a specific region of DNA from
a single DNA molecule even if the starting
material is the whole human genome!
Components of typical PCR
reaction
Template the DNA that contains the target sequence
you want to amplify (1.5-100ng)
Primers synthetic oligonucleotide whose sequence
matches a region flanking the target you want to amplify
and primes the manufacture of a new strand by DNA
polymerase (50-100pmoles of each primer)
Buffer (supplied with the enzyme but typically contains
Tris/HCl (10-50mM), KCl (50mM), Triton-x-100 (0.1%)
Magnesium chloride (0.5-5mM)
dNTPs (deoxynucleotides) (50-200uM of each dNTP)
DNA polymerase, usually Taq polymerase Heat stable
polymerase (0.5-1u)
Which bits of DNA get specifically
amplified?
The segment of DNA to be amplified is
flanked by two primers:
e.g.
5caatttggatagtagccgtatcgatgcgtagtcagatgaggtga
ccagtggatgacgattgacattaagtgaacggtgacccaaaagt
gacgatagacagttgacaaagttgacagtagacgatagacaa
gttgtttggaccaaagtgacacgtgacgttttgcaaacgtgacgtg
aacgttgacacagtgac3


3'
3'
3'
3' 5'
5'
5'
5'
Polymerase Chain Reaction
Target DNA
Denaturation
Extension of
primers
Annealing of
primers
Single-stranded
DNA
Strand separation
Anneal
primers
Extend
primers
95C
50-60C
72C
The PCR reaction has three stages

x~30
PCR is an exponential reaction
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
A
M
O
U
N
T

O
F

D
N
A
PCR in detail
only two
partially double
stranded
products are
produced
denature
Anneal primers
Extend primers
Cycle 1
Round 2 four
partially double
stranded DNAs
are formed but
not the target
sequence
Cycle 2
Products from round 2

Six partially double stranded
products are formed plus two
copies of the target sequence.






At 30 cycles there are
1,073,741,764 target copies
(~1x10
9
). There are also 60
other DNA copies!!
PCR
Gel
electrophoresis
2-3 hours!!
The end result
Checking its worked!
Checked by agarose gel electrophoresis

Is the product the expected size?
Is there more than one product?






Optimising the reaction
From this
To this!!
Optimising the Reaction
Denaturation
Primers can anneal non-specifically during
first round of denaturation.
Polymerase could extend the primers

Use Hot start PCR

Optimising the Reaction
Annealing
The annealing temperature (Tm) is one of the most vital parameters in
a PCR reaction.
Primers have a calculated Tm :

Tm=4(G+C)+2(A+T)
0
C

Annealing temperature chosen depends directly on the length and
composition of the primer
Too low a Tm results in non-specific priming whilst too high affects yield
as the likelihood of annealing is reduced
Usually start with a Tm 5
0
C below the lowest Tm of the primer pair
Annealing happens quickly - ~30sec! Longer annealing times may
produce unwanted products


Annealing
Primer design is vital!!
Primers ideally 18-22 bases long
G/C content should be 45-55%
The annealing temperature (Tm) should
be within 1
0
C of each other. In practice
Tms of 50-80
o
c are common.
The 3 most base should be C or G
Designing Primers
Many web-based design programs
Things to watch out for:
Primers that from hairpins

5 GTTGACTTGATA
T
GAACTCT


-3
Designing primers
Primers that from dimers

5ACCGGTAGCCACGAATTCGT-3
TGCTTAAGCACCGATGGCCA -5

3-
Primer dimers can be a very good, but unwanted
substrate for taq!!!!
Optimising the reaction
Mg
2+
concentration
Mg
2+
is required for:
a)Template-primer stability
b)Activity and fidelity of Taq polymerase

However, dNTPs, template and primers all bind the cation
and limit its availability.
Usual to titrate the Mg
2+
concentration in steps of 0.5mM


Elongation time and temperature
Optimal temperature for Taq is 72
0
C
Will extend at the rate of up to
100bases/second
Rule of thumb: 1kb requires about 1min of
extension time

Paradox how can primers which anneal at
an optimum temperature be elongated at a
considerably higher temperature??
Elongation

Considerations
Taq polymerase:
- is a low fidelity enzyme (errors in base incorporation 1base in 10
4
)
- is denatured by multiple exposures to 94C
- does not work with long templates (>3 kb)

Enhanced thermo-stable DNA polymerases.
- Vent
- Pfu
- Herculase
- Platinum Taq

Cycle Number?
Conc of
product
Cycle number
Increasing the cycle
number above ~35 has little
positive effect. The plateau
is reached when:
- reagents deleted
- polymerase damaged
Plateau
effect
Other things you might find in a
PCR reaction
Helix Destabilisers

Dimethyl sulphoxide
Dimethyl formamide
Urea
Thought to lower the Tm of the target DNA
Care- can also decrease activity of Taq!!


Additives

Glycerol improves amplification of GC-rich sequences
Polyethyleneglycol often used when template concentration is low as
it promotes macromolecular association




Equipment considerations
Thin walled tubes are used for most PCR
applications
Can be bought in 0.2,0.5 or lml format
PCR blocks are also available that take
plates (96well up to 384well)
If possible use a block with a heated lid
Peltier blocks are best because of the fast
heating and cooling (3
0
C per second)
PCR is extremely sensitive
ALWAYS run negative control!!!
Contamination by unwanted DNA can be a
problem
Gloves
Filtered tips
Dedicated areas (UV cabinet)

Whats Wrong With
Agarose Gels?
* Poor precision
* Low sensitivity
* Short dynamic range < 2 logs
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
* Ethidium bromide staining is not very quantitative
ABI: Real-Time PCR vs Traditional PCR (www)
5 copies 50
copies
Fluorescence-based probes
coupled with suitable
instrumentation have
revolutionised quantitative PCR
Real-Time PCR
Real-time PCR monitors the fluorescence
emitted during the reaction as an indicator
of amplicon production at each PCR cycle
(in real time) as opposed to the endpoint
detection
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
A
M
O
U
N
T

O
F

D
N
A
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
A
M
O
U
N
T

O
F

D
N
A
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
Data From a Real-Time PCR Experiment
Log Plot of Real Data
The Log Plot is Linear between 20 and 1500 Units
of Fluorescence
The Threshold Cycle (Ct)
Threshold Cycle
threshold cycle or the C
T
value is
the cycle at which a significant
increase in fluoresecnce is first
detected
C
T
co-incides with the linear phase
of amplification
it is the parameter used for
quantitation

So how do you make the PCR
products fluoresce??
e.gs of chemistry:

DNA Binding dyes (e.g. sybr green)
Hydrolysis probes

Dye Incorporation Assay
* Assays that do not require specificity of probe based assays.
Detection of 1000s of molecules

* General screening of transcripts prior to moving to probe
based assays

* When the PCR system is fully optimized -no primer dimers or
non-specific amplicons, e.g. from genomic DNA
When to choose SYBR Green
Hydrolysis probe (Taqman)
Instrumentation
Instruments on the market:
1) ABI Prism 7900 (Perkin Elmer) (Taqman) can be used for
assays based on DNA binding dyes, Molecular beacons,
Hydrolysis probes.


2) Lightcycler (Roche) can be used with any chemistry
iFluorescence is measured in real-time
3) MyiQ cycler (Biorad) optical module that fits onto their
regular PCR machine - can use any chemistry


Applications of Fluorescence-
Based PCR Technology
Quantitation

Genetic Analysis

Multiplex PCRs
Real-Time PCR for Cancer
Diagnostics
Mutation Detection Using
Lightcylcer Technology
Multiplex PCR
i.e. Screening for multiple genes
simultaneously
Advantages lower reagent costs
speed
Particularly good if tissue is
limited
Disadvantages limited availability of
fluorescent probes
Depends on which Instrument is
available

Advantages of PCR-Based
Diagnostics
- all closed tube systems, minimises hands on time
-- minimal contamination
-- entire process is automated
- Speed
- Cost effective
-- assays easily standardised
-Detection down to a 2x change
-Not much more expensive than conventional PCR
-Requirement for 1000x less RNA than conventional assays

Disadvantages
Assays require skill and competence
High equipment costs
Not ideal for multiplexing depends on
machine
Lability of RNA
Contamination by DNA
References

McPherson MJ, Mller SG (2006) PCR: The Basics.
BIOS, Oxford.

ISBN: 9780415355476
JRUL: Blue, Floor 2: 572.808/MCP

Also electronic resource:
http://www.netLibrary.com/urlapi.asp?action=summary&v=1&bookid=53951


Kleppe K, Ohtsuka E, Kleppe R, Molineux I and Khorana HG (1971) Studies on
polynucleotides. XCVI. Repair replications of short synthetic DNAs as catalysed by DNA
polymerase. J Mol Biol 56: 341-361.

Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N (1985)
Enzymatic amplification of -globin genomic sequences and restriction site analysis for
diagnosis of sickle-cell anaemia. Science 230: 1350-1354.

Saiki RK, Walsh PS, Levenson CH and Erlich HA (1989) Genetic analysis of amplified DNA
with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:
6230-6234.

Mullis KB (1990) The unusual origin of the Polymerase Chain Reaction. Scientific American
262: 56-61.