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POLYMERASE CHAIN
REACTION (PCR)
Bridges 2014
Polymerase Chain Reaction




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Simple reaction
Produces many copies of a specific fragment of
DNA
Live replication takes much longer
Only requires a small amount of DNA (ng)
Many types of PCR
What goes into a PCR?

Mostly water
 Autoclaved

Forward Primer
 Front

end of target DNA sequence
Reverse Primer
 Back

MilliQ
end of target DNA sequence
Template DNA
What goes into PCR?

PCR Buffer
 Balances

MgCl2
 Helps

pH levels
target specificity and stabilizes DNA
dNTPs
 Nucleotides

(A, G, C, T)
Polymerase
 Polymerase
used to “build” target sequence
PCR is SENSITIVE!

Need specific amounts of each reagent
 Too
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Highly subject to contamination
 Use

much or too little can RUIN a PCR
sterile technique!
Needs specific thermocycling program
Thermocycler
Thermocycler program
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
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Denature
Anneal
Extend
Biological Response to Thermocycler
Exponential growth of template DNA
16S rRNA

Ribosomal Sequence
 Ribosomes
make proteins
 All bacteria have 16S

Has conserved and hypervariable regions
 Conserved
regions- primer binding site
 Hypervaribale regions are for species identification