Quantifying DNA in a band on gel electrophoresis

Question

I am a first year science undergraduate and I am just asking this as a more theoretical question rather than for carrying out a protocol, so I would appreciate if answers do not involve lots of complicated chemical names.

I was wondering how one would go about quantifying the amount of DNA in a given band on a gel electrophoresis. Some ideas I have had so far are:

• Cut out the DNA band and extract the DNA. Quantify using some known method such as measuring the absorbance at 260nm of the DNA in solution to find the concentration.
• When the DNA is stained with ethidium bromide, clearly there will generally be more stain uptaken in a band region if it has more DNA so it will fluoresce with greater intensity when illuminated with UV light. However I would have reservations to using this method because I would think that the amount of stain uptaken would depend also on the state of the DNA (i.e. if it is supercoiled or not), but then again I do not know how much of an impact this could have on how much stain is uptaken. Of course, if you know that all of your DNA is in the same state then there shouldn't be a problem with this method I think. Although here you would have to be aware that the concentration of ethidium bromide in a certain band depends on the concentration of nucleotides there, which depends both on the number of the DNA fragments and their size (which is known from the position of the band). Compare this with the final method...
• Finally, if one is making the DNA for example using dideoxy chain termination/Sanger sequencing in PCR, then you can use a radioactively labelled primer. The intensity of the image on the radioautograph would then directly tell you the number of molceules of that DNA fragment present in the band.

I was wondering which of these are viable options to use in the lab, and why/why not.

Answer 1

All three methods could be used to measure the amount of DNA. However in practice, method 2 (estimation by dye brightness) typically works best in a normal workflow. It really depends on what you plan to do as your downstream application.

Problems with method 1:

• Yield from gel purification methods is sometimes finicky and prone to loss.
• Absorbance of remaining agarose can influence the results. DNA cleanup is a must.

Problems with method 2:

• Quantification is difficult as you must estimate the band brightness against a known standard (add a known quantity of your ladder and use that for estimation).
• Supercoiling of plasmids causes multiple bands to appear, so the estimation must be performed multiple times and then added (now it's less of an estimation and more a guess)

Problems with method 3:

• Working with radioactivity adds a significant amount of safety protocols into a simple procedure
• All products from radiolabeling must also be handled as radioactive material
• Using an image has the same issue as method 2 where you're estimating instead of actually quantifying.
• You could get more quantitative results from a scintillation counter, but then the products become more difficult to use because of purification from the scintillation fluid (maybe this is possible but I never tried it).
• You're limited to procedures which would incorporate the radiolabeled nucleotides
• If you do something like dA-tailing, then you're not quantifying the amount of DNA, but the amount of copies of a transcript.