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Biology 224

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Mispriming primers anneal to alternate sites and not to 'correct' or targeted site ... anneal efficiently in 30 sec or less, unless the Ta is too close to ... – PowerPoint PPT presentation

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Title: Biology 224


1
Polymerase Chain Reaction
  • Biology 224
  • Instructor Tom Peavy
  • March 20, 2008

ltFigures from PCR by McPherson Mollergt
2
PCR Polymerase Chain Reaction
  • DNA photocopier
  • integral tool for molecular biologists
  • work horse
  • versatile (many applications)
  • not difficult to perform technically
  • fast

3
  • 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

4
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.)

5
PCR components
Template DNA Primers
dNTPs (water, buffer)
Thermostable polymerase
6
  • Template DNA is denatured (Denaturation phase
    94?C)
  • Primers allowed to anneal to template Tm of
    primers is important (Annealing phase variable
    temperature)
  • Increase temperature to optimum for thermostable
    polymerase (Elongation phase ?68-72?C)
  • Repeat the whole cycle starting at step 1

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8
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
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10
Sources of Template DNA
Genomic DNA RNA isolation and cDNA Plasmid,
bacteriophage, cosmid and artifical
chromosome DNA Pathological and forensic
samples Archaeological samples
11
Template amount
-very sensitive technique (dont need much target
template) but the amount of template is likely
to require optimization (generally lt1 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)
12
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)
13
PCR of non-specific sequences or misprimed
products leads to either smeary gels or
unexpected amplimers sizes
14
Artifactual products on agarose gels can arise
from Primer-Dimer formation
15
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
16
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
17
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
18
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
19
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)
20
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21
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
22
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 (gt
70?C)
23
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)
24
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
25
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 (GC) 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)

26
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.
27
Primer Length
The optimum length of a primer depends upon its
(AT) 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)
28
Primers can be designed with engineered sites at
the 5end (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
29
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
30
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32
For the opposite direction (5 end race) need to
reverse complement the sequence!
5
3
3
5
complement
CGN CTG TGN CTT ACC CTG TTT CCN CTT GTG CCN
A C A C
C A
5
3
reverse
NCC GTG TTC NCC TTT GTC CCA TTC NGT GTC NGC
A C C A C
A
33
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
34
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
3
GTG TTC NCC TTT GTC CCA TTC NGT A C
C A C

(24mer) degeneracy (1/4)2 (1/2)5 1/512
35
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
36
General Rules for Primer Design
  • - primers should be 17-25 bases in length
  • - base composition should be 50-60 (GC)
  • 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.

37
Examples of inter- and intra-primer
complementarity which would result in problems
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39
Real-time PCR quantitation
40
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)

41
Short Tandem Repeats (STRs)
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
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