Estimation of DNA by Diphenylamine method

Hello guys ^_^

Long time since a Chemical estimation done and that is why we will be discussing DNA estimation today. This can be done by two methods, one of which we will be learning today. That is Diphenylamine method (DPA method).

Let's do it.

So, What is the test about?

This test helps in detection of DNA both qualitatively and quantitatively. Plus it is quite easy to carry out. The test is based on the appearance of blue color when deoxyribose sugars react with DPA.

Why does the blue color appears?

Before addition of DPA the DNA sample is boiled which leads to the depurination (Purine bases are removed) and dehydration of the deoxyribose sugars occurs. Due to this DNA converts into a highly reactive form called omega-hydroxylevunylaldehyde (you write omega symbolically) which reacts with DPA giving the characteristic blue color. 

How is the reagent prepared?

DPA is kinda powdery. I mean not kinda, it is indeed powdery. So you need to weigh it 1gm and then dissolve it into 100 ml glacial acetic acid (DPA never dissolves in water or aqueous solutions). Once it is dissolved add 2.5 ml of conc. sulphuric acid.

P.S. Store the reagent in amber colored bottles and always keep it at 2oC - 8oC. Also never add the reagent in the sample when it cold. It will lead to erroneous readings.

What is the absorbance to be used?

The blue color formed is measured at 595 nm.

How is the test carried out?

For qualitative tests, boil the DNA sample directly for 10 mins in boiling water bath. After 10 mins, remove the tubes and cool it down. Once it's back to normal add 3 ml of DPA and observe the blue color at 595 nm.

For quantitative test, you will need a standard sample of DNA with the known concentration. Dilute the sample accordingly and dilute the test samples too with water. Repeat the above procedures and observe the readings at 595 nm.

To determine the concentration of DNA in unknown samples, the method is similar to other colorimetric estimations i.e. Prepare a standard graph of concentration vs absorbance and extrapolate the readings of the unknown samples.

Limitatons?

This test is sadly, not very specific for DNA :(

Also, only 50% of the concentration of DNA is estimated because as mentioned earlier only purine residues are freed and thus only they participate in the reaction. Pyrimidine residues scarcely participate.

Phew, that's all for this one.

Hope this helps.

High six :D

-Staph <3