PCR polymerase chain reactions

What is PCR

Polymerase chain reactions

Lab-based process that allows millions of copies of a target DNA sequence to be synthesised in a few hours

Importance of PCR

Allows amplification of target DNA

• e.g specific locus

Once amplified the DNA is present in sufficient quantities for further studies

How is PCR powerful

Very small amount of template DNA required

PCR produced is used as template for next round

PCR components

DNA samples/template

PCR primers

dNTPs

DNA polymerase

Buffers and water

PRC component- template DNA

Genomic DNA, plasmid/vector, another PCR product

Target can be 60 bp- over 20000 bp

PCR components- primers

Oligonucleotides

Match the primer binding site on target DNA sequence

Short and single stranded

Usually designed to match with one location in the template DNA

PCR competent- dNTPs

A mix of: dATP, dCTP, dGTP, dTTP

Nucleotides

PCR components- DNA polymerase

Catalysis for synthesis of DNA molecules

Often Taq, DNA polymerase- enzyme

Thermostable

Some include inhibitor which binds to active site

PCR components- Taq DNA polymerase

Isolated from thermos aquaticus in 1976

• a thermophlic bacterium

PCR components- Buffers and water

Contains all components from reaction to work

-water added to reach final reaction volume

PCR principles

1) denaturation

2) annealing (primer binding)

3) extension (synthesis)

PCR principle- time and temp

Denaturation - 5-30s about 94 oC

Annealing- 5-30s 50-60 oC

Extension- 5s-10ish 72 oC

PCR principles- exponential amplification

Product from one cycle becomes template for next

Nomenclature

Products = amplicons

PCR in practice

Carried out in a lab

Reaction set up- on ice

Thermal cycling

Gel electrophoresis

Validating PCR

Success determined by gel electrophoresis

Products separated on an agarose gel

• allowing for size and conc of product to be estimated

qPCR

Quantitative PCR

Used to estimate amount of starting template

Uses fluorescent dye, fluorescence is proportional to amount of product

Multilocus PCR

Amplification of multiple targets simultaneously

PCR contamination

Extremely susceptible to contamination

Precautions

• use of DNA pre plastic-wear and reagents

• Gloves worn by user

• Separation of pre-PCR and post PCR areas of lab

• Negative controls

History

Invented in 1983 by Kary Mullins

Introduction of Taq DNA polymerase in 1985

Advances

• water bath to thermal cyclers

• Reduction in reaction volumes and tube sizes

• Thermal cyclers with hot lids to prevent evaporation