Polymerase
Chain
Reaction
Biology 224
Instructor: Tom Peavy
March 20, 2008
<Figures from PCR
by McPherson & Moller>
PCR= Polymerase Chain Reaction
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“DNA photocopier”
integral tool for molecular biologists
work horse
versatile (many applications)
not difficult to perform technically
fast
• Kary Mullis of Cetus Corp invented PCR in 1983 (Klenow fragment of DNA pol I)
• First paper describing the use of Taq polymerase was in 1988 (Saiki et al., 1988)
• PCR patent issues involving Taq polymerase
PCR applications
• Cloning cDNAs and RAPD
• Cloning genes
• Real-time PCR
• PCR mutagenesis
• PCR probe generation for hybridization
• Population sampling and genotyping
• Genomic fingerprinting
RAPD-PCR
multiplex-PCR
PCR-VNTRs
Micro- and Minisatelline repeat-PCR
• Diagnostic PCR (detection of pathogens, GMOs, etc.)
PCR components
Template DNA
Primers
dNTPs
(water, buffer)
Thermostable
polymerase
1) Template DNA is denatured (Denaturation phase;
94C)
2) Primers allowed to anneal to template; Tm of primers
is important (Annealing phase; variable temperature)
3) Increase temperature to optimum for thermostable
polymerase (Elongation phase; 68-72C)
4) Repeat the whole cycle starting at step 1
PCR Kinetics
Early cycles: primers act like probes
searching for complementary
Sequences on template DNA
Mid cycles: amplication process is
is fully underway with an exponential
accumulation of amplimers
Late cycles: reagents are limiting and
Amplification is suboptimal
Sources of Template DNA
Genomic DNA
RNA isolation and cDNA
Plasmid, bacteriophage, cosmid and
artifical chromosome DNA
Pathological and forensic samples
Archaeological samples
Template amount
-very sensitive technique (don’t need much target template)
but the amount of template is likely to require optimization
(generally <1 nanogram of cloned template and up to a
microgram of genomic DNA are used)
-relative amount of target can be increased (e.g. choose cDNA library
where target should be expressed in large amounts)
Technical Difficulties
Mispriming – primers anneal to alternate sites and not to
“correct” or targeted site
Needle-in-a-haystack (Template in limited amounts)
Mismatches allowed internally if annealing temperature
is low (below Tm)
Misprimed PCR products will continue to be amplified
(PCR primers are incorporated into the amplimer at the
terminal end and will thus serve as a perfect match for future
PCR cycles; large amounts of PCR product accumulate
if in it occurs in the early cycles)
PCR of non-specific sequences or misprimed products
leads to either smeary gels or unexpected amplimers sizes
Artifactual products on agarose gels
can arise from Primer-Dimer formation
Contamination Problems
Carry-over contamination
prior PCR products, clones, or samples with DNA in general
(e.g. cell lysates, genomic or plasmid DNA preparations, etc)
can enter the PCR tube and serve as potential template
-most often due to aerosol from pipetteman
Other template contamination can be due to:
- floating debris (circulation/vents)
-laboratory surfaces
-tissue from self or others (e.g. skin, hair)
-solution contamination
Preventative: aerosol-free tips (cotton plugs), UV irradiation,
Designated PCR set-up area, gloves, premixes, aliquots of reagents
Optimization of PCR
To improve specificity:
template quality
optimize concentrations of Mg2+, other ions, primers,
dNTPs and polymerase
efficient denaturation, high annealing temps, and fast
ramping rates
limiting number of cycles and their length
PCR strategies (e.g. touchdown PCR, hot start PCR
nested PCR)
Primer design
Magnesium ions are critical
-Magnesium ion concentration often needs optimization
exists as dNTP-Mg2+ complexes,
interacts with DNA backbone
influences activity of Taq polymerase;
MgCl2 is used in buffer to adjust concentrations
Between 0.5 and 5mM is generally used (1.5 mM common)
Low concentrations tend to have low yields of PCR product
High concentrations tend to reduce the fidelity of
Taq polymerase and lead to amplification of
non-specific products
NaCl or KCl concentrations also can be optimized
To enhance for efficient denaturation, high annealing temps,
and fast ramping rates:
Use quality PCR machines (may not effectively reach
temperatures or takes long time to ramp)
Use thin walled PCR tubes
Use Polymerase with High fidelity
Taq polymerase (from thermophilic bacterium Thermus aquaticus)
= 94 kDa protein with 2 catalytic properties
1. 5’3’ DNA polymerase (elongates 50-60 nucleotides/second)
2. 5’3’ exonuclease (removes nts in front of growing strand)
Recombinant versions of Taq (enhanced for either purification or
performance)
Lacks 3’5’ exonuclease activity
Fidelity of Taq or error rate is:
1 base misincorporation per 104 nucleotides polymerized
for a 400 bp fragment amplied 106 fold (=20 cycles) results in
in about 33% of the products carrying a mutation
(thus should sequence several PCR amplimers to determine
consensus)
Proofreading DNA polymerases
(= those that contain 3’5’ exonuclease activity)
Proofreading ability is due to the capacity of the enzyme to discriminate
between whether the nucleotide at the 3’ OH of an extending
strand is correctly or incorrectly paired with the template strand
Generally these enzymes are even more thermostable and tolerant of
buffer conditions
However, could chew up mismatched 3’ primer ends also (‘nibbling’)
Increases fidelity about 5-12 depending on enzyme
Examples: Vent®, DeepVent®, Tli (Thermoccocus litoralis),
Pfu (Pyrococcus furiosus), 12 fold
Usually leaves blunt ends for cloning rather than overhangs
Hot Start PCR
Used to overcome non-specific annealing of primers and/or
primer-dimer formation prior to the denaturation step
(annealed primers will be extended as temperature
ramps up to denaturation temp)
Cheapest method is to add polymerase after temperature is
is above 70C
Alternatively can use commercial reagents such as Taq that has
an antibody attached so as to prevent polymerase activity
until the antibody is denatured (> 70C)
Touchdown PCR
Used to increase specific PCR products
Annealing temperature is set slightly above the Tm
of the primers in the early cycles (enhances the chances
of specific annealing of primers vs. non-specific)
Annealing temperature is gradually lowered in subsequent
cycles (e.g. 1C every two cycles) until desired lower
limiting annealing temperature is reached
Effect is that the target sequences are preferentially
amplified in early cycles and then are continued to
be amplified exponentially (out competing non-specific
targets)
Nested PCR
Design two outside primers for the
first reaction,
Then use a portion of the first reaction
as template in a second reaction using
Internal ‘nested’ primers
Annealing Temperature and Primer Design
Primer length and sequence are of critical importance in
designing the parameters of a successful amplification: the
melting temperature of a DNA duplex increases both with its
length, and with increasing (G+C) content: a simple formula for
calculation of the Tm is:
Tm = 4(G + C) + 2(A + T)oC
In setting the annealing temperature of PCR reaction:
• As a rule of thumb, use an annealing temperature (Ta) about
5oC below the lowest Tm of the pair of primers to be used if a
good yield of product is desired
• Alternatively, if an increased specificity is desired, one can either
Perform touchdown PCR (high-low anneal temp)
The Tm of the two primers should not be different
because it may never give appreciable yields
of product due to trade-offs (annealing temperature
appropriate for one but not the other)
Can result in inadvertent "asymmetric" or single-strand
amplification of the most efficiently primed product strand.
Note: Annealing does not take long: most primers will
anneal efficiently in 30 sec or less, unless the Ta is too close to
the Tm, or unless they are unusually long.
Primer Length
The optimum length of a primer depends upon its (A+T)
content, and the Tm of its partner (to avoid large differences)
Another prime consideration is that the primers should be
complex enough so that the likelihood of annealing to sequences
other than the chosen target is very low.
Lengths are generally 17-25mers
(rationale: there is a ¼ chance of finding an A, G, C or T in any
given DNA sequence; there is a 1/16 chance of finding any
dinucleotide sequence (eg. AG); a 1/256 chance of finding a
given 4-base sequence. Thus, a sixteen base sequence will
statistically be present only once in every 416 bases
(=4,294,967,296, or 4 billion):
Primers can be designed with engineered sites at
the 5’end (e.g. restriction enzyme sites, mutations)
Mismatches can also be designed internally to
facilitate in situ mutations (change coding sequence or
create restriction sites)
Note: only use
the annealing
portion to
calculate Tm
EcoRI
Degenerate Primers
For amplification of sequences from different organisms, or for
"evolutionary PCR", one may increase the chances of getting product
by designing "degenerate" primers:
Degenerate primers= a set of primers which have a number of
options at several positions in the sequence so as to allow annealing
to and amplification of a variety of related sequences.
Need to examine all the options for particular amino acids with
Respect to their codon degeneracy
For the opposite direction (5’ end race)
need to reverse complement the sequence!
5’
complement
reverse
3’
5’
3’
CGN CTG TGN CTT ACC CTG TTT CCN CTT GTG CCN
A
C
A C
C
A
5’
3’
NCC GTG TTC NCC TTT GTC CCA TTC NGT GTC NGC
A
C
C A
C
A
Design of degenerate
primers based on amino
acid sequencing:
If you do not know where
the peptide regions are
located in the gene,
then need to design
PCR primers in both
directions and try
various combinations
Degeneracies obviously reduce the specificity of the
primer(s), meaning mismatch opportunities are greater, and
background noise increases
Increased degeneracy means concentration of the individual
primers decreases (of which there is only one exact match)
thus, greater than 512-fold degeneracy should be avoided.
5’
GTG TTC NCC TTT GTC CCA TTC NGT
A
C
C A
C
(24mer) degeneracy= (1/4)2 (1/2)5 = 1/512
3’
Can use deoxyinosine (dI) at degenerate positions rather
than use mixed oligos:
dI base-pairs with any other base, effectively giving a four-fold
degeneracy at any postion in the oligo where it is present
This lessens problems to do with depletion of specific single
oligos in a highly degenerate mixture, but may result in too high
a degeneracy where there are 4 or more dIs in an oligo
General Rules for Primer Design
- primers should be 17-25 bases in length;
- base composition should be 50-60% (G+C);
primers should end (3') in a G or C, or CG or GC
(prevents "breathing" of ends and increases efficiency of priming)
- Tms between 55-80oC are preferred;
- runs of three or more Cs or Gs at the 3'-ends of primers may
promote mispriming at G or C-rich sequences (because of stability
of annealing), and should be avoided;
- 3'-ends of primers should not be complementary (ie. base pair), as
otherwise primer dimers will be synthesised preferentially to any
other product;
- primer self-complementarity (ability to form 2o structures such as
hairpins) should be avoided.
Examples of inter- and intra-primer complementarity
which would result in problems:
Real-time PCR quantitation
Multiplex PCR
- uses multiple PCR primer sets to amplify
Two or more products within single reaction
- used for genotyping applications where simultaneous
analysis of multiple markers is advantageous (or statistically
necessary)
- Can amplify over short tandem repeats (STRs)
Short Tandem Repeats (STRs)
AATG
7 repeats
8 repeats
the repeat region is variable between samples while the
flanking regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another