Transcript Slide 1

PCR- Polymerase chain reaction
•PCR is an in vitro technique for the amplification of a region of DNA which
lies between two regions of known sequence.
•PCR amplification is achieved by using oligonucleotide primers.
•These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.
•The oligonucleotides serve as primers for DNA polymerase and the
denatured strands of the large DNA fragment serves as the template.
•This results in the synthesis of new DNA strands which are
complementary to the parent template strands.
•These new strands have defined 5' ends (the 5' ends of the
oligonucleotide primers)
•The oligonucleotide directed synthesis of daughter DNA strands can be
repeated if the new duplex is denatured (by heating) and additional primers
are allowed to anneal (by cooling to an appropriate temperature).
The steps of the PCR reaction:
• Template denaturation
• Primer annealing
• Primer extension
• These steps comprise a single "cycle" in
the PCR amplification methodology. After
each cycle the newly synthesized DNA
strands can serve as templates in the
next cycle.
Optimization of PCR
a) Mg++ - one of the main variables – affects stability of double helix, used to
mimic temperature
b) Template DNA concentration – one template strand of DNA is needed.
to reduce error by Taq DNA polymerase, a higher DNA concentration can be
used
Too much template may increase the amount of contaminants and reduce
efficiency.
c) Enzymes used – choose the appropriate one for the length or degree of
fidelity required
d) dNTP - can use up to 1.5 mM dNTP, want in excess.
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dNTP chelate Mg++.
Excessive dNTP can increase the error rate and possibly inhibits Taq.
Lowering the dNTP (10-50 uM) may reduce error rate
Larger size PCR fragment need more dNTP.
e) primers - up to 3 uM of primers may be used, but high primer to template
ratio can results in non-specific amplification and primer-dimer formation
PCR optimization continued
f) Thermal cycling
• denaturation time can be increased if template GC content is high
(calculate Tm).
• Extension time should be extended for larger PCR products.
• Extension time is also affected by the enzymes used e.g for Taq - assume
1000 base/min
• The number of cycles can be increased if the number of template DNA
molecules is very low, and decreased if high amount of template DNA is
used
g) Additives • Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in
PCR for template DNA with high GC content (they change the Tm of
primer-template hybridisation reaction and the thermostability of
polymerase enzyme).
• 0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC
content and long stretches of DNA (Long PCR ) Betaine is often the secret
(and unnecessarily expensive) ingredient of many commercial kits.
• BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
h) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
Wilsons buffer may work better than the buffer supplied from commercial
sources especially with hard to amplify pieces of DNA
PCR optimization continued
i) Primer design
In designing primers for PCR,consider::
• length of individual primers between 18-24 bases. Minimum of 15 to
be specific
• it is desirable that the two primers have a close melting temperature
or Tm (say, within 5 o C or so).
• if possible, primer sequence should end with 1-2 GC pairs (GC
clamp)
• each primer pair should be tested for primer-primer interactions.
• primer sequences should be aligned with all DNA sequences
entered in the databases (using BLAST programs) and checked for
similarities with repetitive sequences or with other loci, elsewhere in
the genome.
• cycling conditions and buffer concentrations should be adjusted for
each primer pair, so that amplification of the desired locus is
specific, with no secondary products
http://frodo.wi.mit.edu/- Primer3
• Primer3 is a widely used program for designing
PCR primers. Primer3 can also design
hybridization probes and sequencing primers.
• PCR is used for many different goals.
Consequently, primer3 has many different input
parameters that you control and that tell primer3
exactly what characteristics make good primers
for your goals.
PCR methods
•
Hot-start PCR- preparing PCR at room temperature can
generate secondary non specific products in the first PCR
cycle that are amplified in subsequent cycles. Hot PCR
prevents non-specific extension at ambient temperatures by
either excluding or reversibly inhibiting the polymerase
enzyme. Most common way to do this is separate the
polymerase with wax until after the first denaturing step.
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"Touch-down" PCR - start at high annealing temperature,
then decrease annealing temperature in steps to reduce nonspecific PCR product. An annealing temperature that is higher
than the target optimum is used in early PCR cycles. The
annealing temperature is decreased by 1°C every cycle or
every second cycle until a specified or 'touchdown' annealing
temperature is reached. The touchdown temperature is then
used for the remaining number of cycles. This allows for the
enrichment of the correct product over any non-specific
product.
PCR methods cont.
• Nested PCR - use to synthesize more reliable product - PCR using a
outer set of primers and the product of this PCR is used for further
PCR reaction using an inner set of primers. Reduces the
contaminations in products due to the amplification of unexpected
primer binding sites.
• Inverse PCR - for amplification of regions flanking a known
sequence. DNA is digested, the desired fragment is circularized by
ligation, then PCR using primer complementary to the known
sequence extending outwards. (see next slide)
• AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic
DNA) - methods for creating genomic fingerprints from species with
little-known target sequences by amplifying using arbitrary
oligonucleotides. It is normally done at low and then high stringency
to determine the relatedness of species or for analysis of Restriction
Fragment Length Polymorphisms (RFLP).
Inverse PCR
PCR methods cont.
• RT-PCR (reverse transcriptase) - using RNA-directed DNA
polymerase to synthesize cDNAs which is then used for
PCR and is extremely sensitive for detecting the expression
of a specific sequence in a tissue or cells. It may also be
use to quantify mRNA transcripts.
• RACE (rapid amplificaton of cDNA ends) - used where
information about DNA/protein sequence is limited. It allows
amplification of an unknown end portion of a transcript using
known information from the centre. It can be used to amplify
3' or 5' ends of cDNAs generating fragments of cDNA with
only one specific primer each (+ one adaptor primer).
Overlapping RACE products can then be combined to
produce full cDNA.
PCR methods cont.
• Multiplex-PCR - 2 or more unique targets of DNA sequences in the
same specimen are amplified simultaneously. E.g. One can be use
as control to verify the integrity of PCR. Can be used for mutational
analysis and identification of pathogens.
• Asymmetric PCR –results in synthesis of mainly ssDNA by the most
abundant primer. Useful for making probes or primers.
• In Situ PCR – done on a slide to identify where in a section or cell a
certain transcript or piece of DNA is located
• Mutagenesis by PCR – used to introduce mutations using the
primers