Amplification of DNA using the
Polymerase Chain Reaction
(PCR)
Polymerization of nucleotides by DNA polymerase
Phosphodiester bonds are formed by a nucleophilic attack of a free 3’OH on the 5’-a-PO4 of the incoming dNTP.
Requirements for DNA replication
The enzyme DNA polymerase.
A DNA template to guide synthesis.
A primer, a short nucleotide segment complementary to the
template that can provide a free 3’-OH for synthesis.
Replication by E. coli DNA polymerase III
E. coli DNA polymerase III beta subunit has a doughnutshaped hole lined with positively-charged amino acid side
chains that interact with the negatively-charged DNA strand
DNA polymerase III  subunit three-dimensional structure
Annual Review of Biochemistry (1995) 64, 176-200. PDB ID 3BEP
Thermus aquaticus (Taq) DNA polymerase
DNA polymerase isolated from a bacterium found in hot springs
Can withstand the high temperatures in PCR needed to denature
double stranded DNA templates
Can replicate 1000 base pairs of DNA in 10 seconds at 72°C
Has low replication fidelity; error rate of 1 in 9,000 nucleotides
A variety of thermostable polymerases that have a greater fidelity are
now available from several companies.
Thermal cycling during PCR
Initial denaturation
Cycle 1
Extension
Annealing
Cycle 2
Cycle 3
Cycle 30
Primer Design for PCR
Optimal length for PCR is 18–30 nucleotides
Percentage of dG or dC should be between 40% and 60%
Avoid complementarity of two or three bases at the 3' ends of primer
pairs to reduce primer–dimer formation
Avoid mismatches between the primer and the target-template
sequence, especially at the 3' end of the primer
Avoid a 3'-end dT. Primers with a dT at the 3' end have a greater
tolerance of mismatch and may bind to sequences other than the
desired sequence.
Use a final concentration of 0.1–0.5 μM (pmol/µl) of each primer.
Primer concentration of 0.2 μM is usually sufficient.
Primer-Dimers
Formed due to self-priming by
one or both primers
Avoid complementary 3’ ends
Each primer-dimer formed serves
as a template in the next cycle
Melting temperature
The melting temperature (Tm) of a primer is defined as the
temperature at which half of the oligonucleotide forms a stable
double helix with the template DNA and the other half is separated
into single stranded species. There are many methods for calculating
the melting temperature of a primer-template pair, some very
complicated. A simplified formula for estimating melting
temperature for an oligonucleotide, up to 20 nucleotides and
assuming no mismatched base pairs, is
Tm = 2°C x (A+T) + 4°C x (G+C)
Optimal annealing temperatures may be above or below the
estimated Tm. As a starting point, use an annealing temperature 5°C
below the calculated Tm.
Melting temperature
A more accurate formula that can be used for oligonucleotides
between 15 and 70 nucleotides is
Tm = 81.5 + 16.6(log(I)) + 0.41(%G+C) – (600/N)
Where I is the molar concentration of monovalent cations and N is
the length of the oligonucleotide.
Another formula that can be used for oligonucleotides between 20
and 35 nucleotides is
Tm = 22 + 1.46 ((2 x G+C) + (A+T))
DNA polymerases used for PCR
Two classes of DNA polymerase are commonly used according to
the template they copy
DNA-dependent DNA polymerases
RNA-dependent DNA polymerases (aka reverse transcriptases)
Two properties are often considered
Processivity –affinity of the enzyme for the template
Fidelity – accuracy of synthesis and a measure of the error rate
See http://www.invitrogen.com/site/us/en/home/Products-andServices/Applications/PCR/pcr-enzymes-master-mixes.html for an
example of choosing a PCR enzyme
Commercial Sources of Taq DNA polymerase
Amplitaq® - Perkin Elmer Applied Biosystems
Recombinant protein produced in E. coli
Purified using a heating step
DNA possibly present in the sample may contaminate PCR reactions
Amplitaq® LD (low DNA) or native Taq can be used
Amplitaq Gold®
Taq is chemically modified and inactive until heated at 94ºC for 10
minutes (hot start)
Traditional hot start used antibodies for inactivation
Stoffel fragment of Taq
Lacks 289 N-terminal amino acids of Amplitaq
Lacks 5’-3’ exonuclease activity of Amplitaq
2-fold more thermostable that Amplitaq
Fidelity of Taq DNA polymerase
Taq lacks a 3’-5’ exonuclease “proofreading” activity.
Taq will incorporate 1 error in 1000 nucleotides
Taq will cause a frameshift 1 in 4000 nucleotides
Many labs now use thermostable proofreading enzymes that contain
3’-5’ exonuclease activity for PCR reactions
Note: Taq DNA polymerase
preferentially adds an adenine to
the 3' end of the product. Such PCR
amplified inserts can be cloned into
linearized vectors that have
complementary 3' thymine
overhangs.
Properties of some thermostable DNA polymerases
Source: McPherson and Møller. PCR The Basics. Taylor and Francis 2006. Page 38.
Polymerase Mixtures
The use of a mixture containing
predominantly Taq with a low
concentration of a proofreading enzyme
improves fidelity but allows high levels of
product to be formed.
Often called “long-range” PCR
Advantage®2 mix from Clontech
Expand™ High Fidelity PCR system from
Roche contains Taq and Pwo DNA
polymerases
PCR additives or enhancers
Certain PCR templates are difficult to amplify, often due to high GC
base content. Chemical additives, such as
dimethylsulfoxide (DMSO), up to 10%,
or formamide, up to 5%, reduce
secondary structure and base pairing.
Other additives are:
Trimethylammonium chloride (10-100 uM)
Betaine (N,N,N-trimethylglycine) 1-1.3M
Nonionic detergents such as Tween 20 at 0.1-2.5%
Polyethylene glycol (PEG) 6000 5-15%
Glycerol 10-15%
Many DNA polymerases purchased commercially come with PCR
additives or enhancers of unknown identity.
Control reactions and optimization of PCR
Control tubes should be run with:
No DNA
No primers
Single primers
The following will need to be optimized:
Magnesium ion concentration
Other ions (At KCl>0.2M DNA denaturation is inhibited)
DNA polymerase concentration
Temperatures (denaturation, annealing, extension)
Cycle number and length (kept to a minimum)
Template concentration (may have to dilute contaminants)
Touchdown PCR
Starts initially with an annealing temperature higher than the Tm of
the primers and then gradually decreases to below the Tm.
This ensures that only specific annealing of the primers to their
correct target sequence occurs before any nonspecific annealing
events.
Example:
Over first 20 cycles start at 65ºC and reduce annealing temperature
1ºC every 2 cycles to 55ºC. Then run 10 more cycles at 55ºC.
Hot start PCR
Specificity problems can arise before the first cycle of PCR. Nonspecific
annealing can occur before initial denaturation.
Could wait until PCR reaction is at 95ºC before adding DNA polymerase. That is
not practical.
The most common hot start is to
inactivate DNA polymerase until the
95ºC denaturation temperature is
reached. Inhibitory monoclonal
antibodies that bind to the polymerase
and inhibit are denatured and released at 95ºC.
Wax beads can encapsulate DNA polymerase. The wax melts at high temperature
and releases the enzyme. (Taq Bead hot start polymerase from Promega)
Magnesium also has been encapsulated in wax beads.
Nested PCR
If PCR produces a mix of
desired and undesired
products, PCR1 DNA can be
used in a second PCR with
primers internal to the primer
binding sites from the first
PCR reaction.
The odds of undesired PCR
products containing both
primer binding sets is
essentially zero.
RT-PCR
RT-PCR utilizes a reverse transcriptase to produce a complementary DNA (cDNA) product from an
RNA template
RT-PCR systems purchased commercially typically contain a reverse transcriptase (like Tth
polymerase) and DNA polymerase(s) (like a mixture of Taq and Pwo DNA polymerases)
Reverse transcriptases have two activities:
DNA polymerase activity which requires a RNA or DNA primer is required to initiate synthesis.
RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DNA hybrids, such as
are formed during reverse transcription of an RNA template.
RT-PCR, is a useful tool for such things as diagnosing microbial diseases rapidly and a myriad of other
applications. In many cases, standard preparations of reverse transcriptase are used for RT-PCR, but
mutated forms with relatively high thermal stability have been developed to facilitate the process.
See http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/reversetranscription/reverse-transcriptase-enzymes.html for an example.
Standard RT-PCR
Reverse transcription from mRNA using an oligo-dT primer.
Primers can also be specific for a certain gene.
Semi-quantitative RT-PCR
Differences in mRNA levels in samples can be estimated using RTPCR. The amount of PCR sample produced during the exponential
stage of amplification is used for analysis. Primer pairs and reaction
conditions must be identical.
Lanes 1&2 – A. thaliana flowers
Lanes 3&4 – A. thaliana roots
Lane 1 – 10 cycles of PCR
Lane 2 – 15 cycles of PCR
Lane 3 – 10 cycles of PCR
Lane 4 – 15 cycles of PCR
Quantitative
RT-PCR
Coamplification of both the
target mRNA and a standard
mRNA in a single reaction,
termed competitor PCR, is
used to quantify mRNA
levels in a sample.
The competitor is designed
to have the same primer
binding sites.
Various known amounts of
competitor RNA is mixed
with the sample containing
the target RNA.
Quantitative real-time PCR (QPCR)
QPCR is used to quantify the amount of a specific DNA present in a sample, often
viral or bacterial in origin, or human blood or tissue. The target DNA may be
present at high or low levels.
During QPCR, the amount of PCR product formed is measured each cycle and
reported in fluorescence units. The more target DNA present in a sample, the more
quickly the PCR product (and therefore fluorescence) is generated.
Threshold Cycle
The threshold cycle (Ct) value denotes how many cycles of PCR are
required for the amount of PCR product (measured by fluorescence)
to reach a defined threshold value. The more target DNA present in
a sample, the lower the Ct value will be, as the threshold is reached
sooner.
Fluorescent detection
Detection of DNA product formed (amplicon) utilizes a molecule such as
SYBR green that binds to double-stranded DNA or a TaqMan probe.
The fluorescent dye SYBR Green
binds to the minor groove of the
DNA double helix. In solution, the
unbound dye exhibits very little
fluorescence, however, fluorescence
is greatly enhanced upon DNA
binding.
TaqMan assays exploit the 5'exonuclease activity of Taq. The
probe is labeled with a fluorescent
reporter at the 5' end, often
fluorescein (FAM), and a quencher
dye at the 3' end.
(www.bio-rad.com)
Amplification of DNA using PCR
Reagents:
Taq DNA polymerase
Amplification buffer
Magnesium chloride (MgCl2)
deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
Template DNA (E. coli genomic DNA)
5’ primer (100 mM concentration)
3’ primer (100 mM concentration)
H2O to 50 ml volume
Thermocycle parameters:
1. 94ºC for 5 minutes
2. 94ºC for 15 seconds
3. 45ºC or 55ºC for 30 seconds
4. 68ºC for 1 minute
5. Go to step 2, repeat 29 times
6. 4ºC forever
Plan your PCR reaction