DNA amplification- quantification

Reasons for quantification 1 

• If too little DNA is input into a PCR reaction

• Fail completely

-trace- little to no DNA present 

-poor storage- decay

-poor csi training 

• Need repeating- expensive

• Result in low signal to noise- difficult to interpret

Reasons for quantification 2

• If too much DNA is input into a PCR reaction

• Fail completely

-too much DNA can inhibit PCR

-usually (not always) occurs if quantification step missed

• Need repeating

• Result in high signal to noise- difficult to interpret

Methods of quantification- agarose gel visualisation

• Simple way to confirm DNA is present- low accuracy

• Does not give a concentration- low precision

• Does not provide any information on inhibitors

• Stained with an intercalating dye

What is UV- visible spectrophotometry used for

To understand the concentration of something

-does not waste precious sample

-is cheaper per sample

-system is very easy to use

Limitations to UV- visible spectrophotometry 

• Its not a sensitive approach 

• limit of detection is -2ng/ul

• what about forensic samples ?

Real time analysis

• mid 1990s people began to think about collecting the data during the PCR- qPCR or real time PCR

• early work involved mounting a fluorescent camera above the PCR machine and pipetting ethidium bromide directly into thhe PCR wells

• specific machines have been developed since and the approach is commonly used