PCR- Polymerase chain reaction

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• 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).

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• 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.

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• 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.
• 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

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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

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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

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• 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.

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• 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.
• "Touch-down" PCR - start at high annealing
  temperature, then decrease annealing temperature
  in steps to reduce non-specific PCR product. An
  annealing temperature that is higher than the
  target optimum is used in early PCR cycles. The
  annealing temperature is decreased by 1C 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.

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• 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).

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Inverse PCR
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• 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.

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• 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

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