DNA amplification- how its done

Who discovered the PCR?

Kary mullis in 1983

What are the things needed for PCR

• Thermal cycler

• Template DNA

• PCR primers

• DNA polymerase- enzyme

• PCR buffer

Template DNA

• Dna needs to be present

• Ideally single sourced

PCR primers- how many are used

• Two primers- used to amplify a single DNA target region

What are the primers termed as 

Forward and reverse primer

What are the primers designed to do

• Flank the target region

• Designed to be highly specific to the target DNA

What is the size of the DNA fragment produced in PCR dependent on

The primer locations (and any mutations that occur)

DNA polymerase and buffer components- what is the DNA polymerase known as 

A biological catalyst- the PCR needs this 

DNA polymerase and buffer components

• DNA polymerase can only add free nucleotides to the 3’ end of the chain

• PCR requires nucleotides without a O on the 2’- deoxynucleotide triphophate (dNTP)

What is there large amounts of in the PCR buffer 

Large amounts of each dNTP

• dATP, dTTP, dCTP, dGTP

What is the by product of the reaction

A pyro-phosphate

What are additional buffer components in PR that facilitate the reaction

MgCI2

Tween

BSA

What is MgCI2 needed for

The incorporation of dNTPs into the new strand 

What is tween used for

Stabilises polymerase and lyses cells 

What is BSA used for 

Acts to prevent inhibition

Prevents adhesion of enzymes to the reaction tubes and tip surfaces 

Cycle number- why do we stop amplifying

• To prevent signal saturation

• Ambiguous results may be challenged

• To reduce the formation of non-specific products

• Usually formed by sub-optimal PCR conditions, poor primer design

• Risk of contamination is greater with more cycles

PCR inhibitors

• Forensic samples often contain impurities

• Recovered during collection- environmental, biological and chemical

• Most removed during DNA extraction