Question

Why do we use a negative control in PCR?

Answer 1

See below

Explanation:

PCR works off of a template DNA. Lets say you are testing for HIV (HIV is an RNA virus, but when it goes into a cell, it gets turned into DNA....so there will be HIV DNA in an infected cell). The primers you use will make a product (amplicon) that corresponds to part of the HIV DNA. If you see this amplicon, then you have the HIV sequence present.....but if you don't have a negative control, you may have contamination.

PCR is extremely senstitive. There are many solutions used in PCR (water, buffer, dNTPs, enzyme)...and all of them can easily get contaminated with DNA from other samples, or even from the amplicon that was made in the reaction you did yesterday. So if you have Patient X's sample of DNA and you are checking for HIV by PCR, the PCR primers may make a product off of the HIV DNA in Patient X's DNA sample (if that person has HIV), or it may make it off of contamination. But you can't tell if it is from contamination or from HIV in the patients DNA.

So you run a water control. Tube 1 you place all the components of the reaction, and for the DNA you only add water. This is the negative control. NOTHING should amplify here. In Tube 2 you put all the reaction components and Patient X's DNA. If you get a product here, (and nothing in Tube 1), Patient X probably has the HIV DNA in his/her DNA. If you get a product in both Tube 1 and Tube 2, you have a contamination problem and you can't tell if the HIV in the Patient's sample is from the disease or from contamination.

So you always run a water control (water in place of DNA).