04. PCRin vitro enzymatic amplification of DNA

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04. PCR in vitro enzymatic amplification of DNA
- From Kleppe Khorana to Kary Mullis
gt from repair synthesis to exponential
amplification via synthetic oligonucleotides -
basic concept of amplification when product
becomes substrate the PCR cycle denaturation,
annealing, elongation (2n - 2n) amplicon
molecules

2

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correctly
A 2n - 2n 1. 2 - 2 0 2. 4
- 4 0 3. 8 - 6 2 4. 16 -
8 8 5. 32 - 10 22 6. 64 - 12
52 7. 128 -14 114 8. 256 -16 240
9. 512 -18 494 10. 1024-20 1004 ... 20.
1,048,536 ... 30. 1,073,741,764
incorrect estimation

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Properties of some early thermostable DNA
polymerases
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Properties of Taq polymerase - single
polypeptide chain of 94 kDa 200,000 U/mg -
Topt 70 - 80 C - Kcat up to 150 nt per
second per enzyme molecule (at 55C still
about 24 nt/s) - no 3' gt 5' exo activity, but
has 5' gt 3' exo activity (gt
misincorporation frequencies 10-5 to 2x10-4 per
nt per cycle) - terminal transferase activity
adds an extra A (or other nt) at 3' ends (an A
if all four dNTP are present with a unique
dBTP, addition of the B) - large diversity of
templates, only minimal amounts required in
situ lysis of E.coli, B.subtilis, Streptomyces,
yeasts, animals cells or plant protoplasts may
be sufficient also fossile material,
microscope slides, blood stain, etc)
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PCR fragments with an extra protruding A at the
3' end can be cloned by T/A cloning. Vector may
be prepared by blunt end cleavage, followed
treatment with Taq polymerase dTTP (solely) to
add an extra T at the 3' ends. Alternatively,
the vector may contain two HphI recognition sites
in anti-parallel orientation, so that upon
cleave a single T residue remains single-stranded
at each 3' end (see hereunder)
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Cloning of PCR fragments by creating sticky ends
with 5' extended primers followed by restriction
cleavage.

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Temperature profiles of PCR reactions in
different devices. Internal tube temperatures.
(a) three water baths with robot arm (b e)
programmable heating/cooling blocks Cycling
of 45C, 72C, 93C
Ramping times are important. Polymerase remains
active during the intervals.
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General aspects - problem of sensitivity
versus risk of contamination - size range of
amplicon 200-2000 bp versus 2 - 5 kb
versus 5 - 50 kb (long-range PCR) -
amplification process - temperature profile
machine-dependent - touch-down PCR start
too high, decrease 0.5C per two cycles -
hot-start procedure (temperature or
antibodies) hot start omit one crucial
component until temperature above annealing
temperature is reached alternative anti-Taq
antibody (reversible inactivation)
(inactive) antibody-polymerase complex that
dissociates at denaturing condition - efficiency
A E(n).2n Nf No (1 E(n))n Em
(Nf/No)1/n -1 amplification numbe
r of molecules mean efficiency per cycle -
decreasing efficiency at 1012 "targets" (or even
less) - increasing amplicon length
"long-range" PCR - go across defects -
use mixture of polymerases
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- primer choice - size, GC (40-60,
balanced between both primers) - avoid
complementarity between primer ends, and within
primer - mismatches, tails Basic goals -
analytical (identify presence of sequence, in
particular if only minute amounts are present)
- synthetic (increase amount of DNA fragment,
make new combinations, etc...) examples of
applications - clone analysis (replacing
plasmid isolation restriction analysis) -
screening genetic errors (diploid gt homozygote,
heterozygote) (including prenatal) -
screening for viruses and other pathogens (e.g.
HIV, HCV, etc.) - diagnosis of bacterial
infections medical, veterinarian, food industry
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HhaI polymorphism close to the cystic fibrosis
locus of chromosome 7. Samples (0.5 mg) of
genomic DNA were amplified for 30 cycles,
digested with HhaI, and fractionated in agarose
gel. Visualisation of the fragments by UV
fluorescence after ethidium bromide
staining. The polymorphic restriction site is
central. When the site is present, the 330 bp
fragment is cleaved into two 165 bp fragments.
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Variations - nested PCR - RT-PCR (use
of reverse transcriptase, use of Tth
polymerase) - A-PCR asymmetric PCR -
inverse PCR, RAcE (see chapter on DNA
libraries) - (semi-)quantitative PCR,
competitive PCR, real-time PCR - SOE (see
also chapter Directed mutagenesis) -
incorporation of nucleotide analogs and tagging
(see earlier) - LIC 'ligation independent
cloning' (not a PCR technique, but
the fragments to be cloned usually prepared by
PCR) (see Chapter 2 Enzymes gt
T4 polymerase)
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Nested PCR purpose increase
sensitivity while maintaining accuracy

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Asymmetric PCR Generation of single-stranded
DNA
Main application preparing templates for DNA
sequencing
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Inverse PCR Amplification for sequencing outside
the boundaries of a known sequence (shaded).
R1 (unpredicted) and R2 (predicted) are
restriction sites. A (very) complex mixture is
formed, but after self-ligation (circularization)
only the region flanked by primers a and b in
opposite orientation are amplified by PCR.
nb. self-ligation requires low DNA concentration
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SOE splicing by overlap extension
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Real-time PCR DNA amplification plot, based on
fluorescence of ethidiumbromide (bound to dsDNA).
At cycle number CT, the fluorescence exceeds
the threshold (set closely above the background)
Background is high because the intrinsic
fluorescence of ethidiumbromide. This may be
improved by the use of SYBR Green I. But in
both cases, the problem remains that they bind
not only to the proper ds-PCR product but also to
non-specific products, primers, etc. Therefore,
new procedures use probing systems, based on
fluorescent tags, including quenching. (see
slides below)
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Initial DNA concentration versus DNA yield,
monitored by increase of fluorescence
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Calculating the efficiency (E) of a PCR reaction.
CT values versus the logarithm of the initial
DNA concentration (see previous Figure)
The logarithm of the initial target copy number
versus CT gives a straight line with a slope of
-1/logE (E efficiency, maximum
efficiency 2)
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Real-time quantitative PCR Release of reporter
proportional to the amount of amplicon
produced (cleavage of the probe)

The use of a set of different fluorophores
(different colors) allows to test different
targets in parallel (if they have the same
amplification efficiency)
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Molecular beacon Quenching of fluorescence if
the stem-loop structure holds together.
Binding to the target separates the helix and
the fluorophore is separated from the
quencher. (no cleavage of the probe)
second order kinetics
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The use of Scorpion probes
Intramolecular hybridisation of the the extended
sequence disrupts the quenching of
the fluorescence.
first order kinetics (faster)
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LIC Ligation-independent cloning - Vector,
cleaved by SmaI fragment 1 - DNA fragment
for insertion fragment 2 prepared by PCR
using primers with 5' tails that match the
vector ends (see figure).
fragments cannot self-circularize.
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Addendum LCR ligase chain reaction based
on the same concept as PCR a technique of
cyclic ligase reaction (with thermostable ligase)
for use in diagnosis (e.g. of mutants) gt
"amplification" of ligated oligonucleotides appl
icable only with ligases devoid of blunt-end
ligation activity
p
p
p
p
Accumulation of ligated oligonucleotides if
perfect complementarity at the nick. The
product from each ligation reaction becomes
(extra) substrate in the next cycle.