PCR

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The PCR - amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence.

These are extended towards each other by a thermostable DNA polymerase in a reaction cycle –

Three steps:

Denaturation

Primer annealing

Polymerisation/elongation/extention

PCR is DNA replication in a test tube: PCR uses just one indispensable enzyme – heat resistant DNA polymerase – to amplify a specific fraction of the genome. High temperatures denatures DNA to single strands.

Sequence of the DNA of DNA/RNA must be known e.g. which organism are you looking for. Cannot ask for PCR without indicating the name of the organism.

Primers are 20 – 22 nucleotides long. Have a target sequence of 100 – 1000 bp (with 5000 as practical limit).

Have 10 – 12 Gs or Cs. They form 3 hydrogen bonds while As and Ts only have 2.

PCR products can be used as templates to sequence genes

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Other types of PCR

Multiplex:

More than one reaction/primers in the same tube

NB - Annealing temperature of primers must be in the same range

Detection of multiple products in one tube

Nested PCR:

2 set of primers: Larger product, Smaller product.

Second set of primers used on the first amplicon – shorter amplicon

Uses: e.g. increase sensitivity, to distinguish between species within a genus

RAPD PCR - Random amplified polymorphic DNA:

Use short random primers

Lower annealing temperature

Non-specific binding and amplification

Analysis and interpretation difficult

Microsatellites:

Use short tandem repeats 2, 3 or 4 bp – DNA fingerprinting

In situ PCR:

•PCR carried out in cells or fixed tissue on a slide - histology

Radio/fluorescence labelled PCR product use as probe to detect target

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Methods of PCR:

Real-time PCR (Quantitative PCR):

Detect amplification at each cycle

Monitor amplification online and in real-time

Quickly and accurately quantify results by either

Taqman Probe Principle:

Probe

Contains both fluorophore and quencher

Extension removes fluorophore

Taq DNA polymerase 5’ to 3’ exonuclease activity

Permits fluorescence

LAMP PCR:

Loop mediated isothermal amplification of DNA

Robust – ‘field PCR’

PCR done at a single temperature

Use more than 2 primers - multiple

Use intercalated dyes – measure e.g. fluorescence

Amplification in loops