Amplification of DNA using the Polymerase Chain Reaction (PCR)

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Amplification of DNA using the Polymerase Chain
Reaction (PCR)
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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.
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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.
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Replication by E. coli DNA polymerase III
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E. coli DNA polymerase III beta subunit has a
doughnut-shaped hole lined with
positively-charged amino acid side chains that
interact with the negatively-charged DNA strand
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DNA polymerase III ? subunit three-dimensional
structure
Annual Review of Biochemistry (1995) 64, 176-200.
PDB ID 3BEP
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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 72C 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.
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Thermal cycling during PCR
Initial denaturation
Cycle 1 Cycle 2 Cycle 3
Cycle 30
Extension
Annealing
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Primer Design for PCR
Optimal length for PCR is 1830
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 primerdimer 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 0.10.5
µM (pmol/µl) of each primer. Primer
concentration of 0.2 µM is usually sufficient.
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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
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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 2C x (AT) 4C x
(GC)   Optimal annealing temperatures may be
above or below the estimated Tm. As a starting
point, use an annealing temperature 5C below the
calculated Tm.
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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(GC)
(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 GC)
(AT))
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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
-and-Services/Applications/PCR/pcr-enzymes-master-
mixes.html for an example of choosing a PCR enzyme
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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
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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.
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Properties of some thermostable DNA polymerases
Source McPherson and Møller. PCR The Basics.
Taylor and Francis 2006. Page 38.
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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 Advantage2 mix from Clontech Expand High
Fidelity PCR system from Roche contains Taq and
Pwo DNA polymerases
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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.
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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 KClgt0.2M 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)
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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.
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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.
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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.
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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/Applica
tions/PCR/reverse-transcription/reverse-transcript
ase-enzymes.html for an example.
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Standard RT-PCR
Reverse transcription from mRNA using an oligo-dT
primer. Primers can also be specific for a
certain gene.
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Semi-quantitative RT-PCR
Differences in mRNA levels in samples can be
estimated using RT-PCR. 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 12 A. thaliana flowers Lanes 34 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
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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.
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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.
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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.
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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)
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Amplification of DNA using PCR
Reagents Taq DNA polymerase Amplification
buffer Magnesium chloride (MgCl2) deoxynucleo
tide 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