Detection of DNA

[Sukhpreet]

I extracted dna from my cheek cell by using the rubbing alcohol and soap and salt. Now i need an indicator that tells that there is presence of DNA. Is diphenylamine will react with it without any further chemical modification?

[deleted-140482]

Hi,

Can I ask why you need to use an indicator for the presence of DNA? Most experiments of this sort use enough cheek cells (or strawberries, or onions) that the DNA is readily visible to the naked eye and can be collected in a "spool."
See these procedures:
https://www.sciencebuddies.org/science- ... #procedure
https://www.sciencebuddies.org/science- ... #procedure
https://www.sciencebuddies.org/science- ... #procedure

In the lab, we use several methods of identifying/staining DNA. The simplest, crude method is simply to use a UV spectrophotometer, which passes light through the liquid at 260 and 280nm. Using a simple equation, we can calculate the concentration of the DNA as well as the purity (pure DNA will give a 260/280 ratio of 1.. We also often run DNA on a gel and stain with ethidium bromide, a fluorescent dye that binds to DNA. There are a number of other dyes that can be used as well, but none of these procedures is going to be easy to do at home. My understanding is that diphenylamine can be used as a DNA indicator because it reacts with the deoxyribose sugar under acid and heated conditions, but I've never done this myself, so I'm not sure how sensitive it is. I'm also not sure whether diphenylamine is something you should be handling at home without proper safety precautions, so I'd approach this very carefully.

Maybe another expert can chime in with some suggestions, but it would also be helpful if you could provide us with a little more information about precisely what you are trying to do and whether everything needs to be done at home, or if you have a lab of some sort available (even a high school one might be helpful).

I hope this is helpful!
JMP

[Sukhpreet]

Actually, yes there is a lab available for my work but not have expensive devices. My project is to detect DNA without making a spool. My project is about extract dna from very small amount of cheek cells and then to know if there is DNA present in water or solution. and can u please a method of detecting DNA. The amount salt water that rinse in my mouth(in which cheek cell present) is about 20ml and there is small threads but i have to show that this is a DNA with any test.
With kind Regards

[SciB]

Hi,

Diphenylamine reacts with the deoxyribose sugar of DNA to form a blue color but it also reacts to a lesser extent with RNA. This is an old test for DNA that is rarely used anymore. The best way to tell how much DNA is in your solution is to use a UV spectrophotometer and measure the solution's absorbance at the wavelengths of 260 nm and 280 nm. DNA and RNA absorb strongly at 260 nm and protein absorbs at 280 nm.

If you have to use the diphenylamine test, you will need to do it in a lab with supervision as the preparation of the diphenylamine reagent solution requires glacial acetic acid and concentrated sulfuric acid, both extremely hazardous to humans. I would recommend that you ask around at a university to see if you can use a spectrophotometer to test for DNA. You will need some of the salt solution BEFORE you rinsed your mouth with it to use as a blank (zero DNA). Reading the 260/280 values of a DNA solution is a very simple procedure and takes only a couple minutes. People in molecular biology labs who are doing cloning do this frequently and most would be happy to help a high school student read their DNA solution.

If you have any more questions, please post back.

Sybee

[Sukhpreet]

Thanks this is really helpful. Lets say that water have some bacteria in it. Does it affect the absorbency ? to set on zero salt water used or salt+water+alcohol used?

[SciB]

Bacteria have their own DNA, so yes they could affect the reading but i don't think there's enough of them to be noticeable. For the blank you need to use the same solution that you used to rinse the cheek cells from your mouth. Did you add anything to this solution after you spit it out? If you did, then that needs to go into the blank also. The blank needs to be identical to your actual sample except for not containing cheek cells. The UV spectrophotometer reads the absorbance of the blank at 260 nm and subtracts it from the reading of your sample so the difference has to be due to your cells.

There's a formula for calculating the approximate concentration of DNA from the A260 reading: http://homepages.bw.edu/~mbumbuli/molbio/labs/dna/

Also, once you have the DNA concentration and you know the volume of the solution that you used to rinse your mouth, you can determine the approximate number of cheek cells that you recovered by assuming that each cell contains about 6 pg (picograms) of DNA. There are one billion pg in a milligram (mg) so calculate how many mg of DNA you have in your sample and multiply that by one billion and divide by 6 to get the number of cells. So, now you know how many cells you lose every time you rinse your mouth out!

Let us know if you have more questions.

Sybee

[Sukhpreet]

Thank you. lets say i take 1 ml of rinse solution and the OD is 0.55 what that means? how i convert it into number of cells?

[deleted-143835]

Hi Sukhpreet,

OD, which stands for optical density, is like a ratio to express the concentration of cells in solution. So 0.55 is a relative measure in a sense but can offer valuable information about how concentrated or dense the solution is. I believe there is a formula to convert between OD values to actual cell count; however, this likely varies from species to species of bacteria. Try a Google search: just for your reference, here is the calculator for E. coli: http://www.genomics.agilent.com/biocalc ... tid=290172

Although, from my microbiology experience, it usually suffices to express concentrations in terms of OD (scientifically sound) rather than cell count in numbers. It depends if your project really specifically requires the cell count value or not!

Hope this helps,
scibuddyAK

[Sukhpreet]

Ok. Lets say i have 3 e-oli bacteria and i incubate them in the LB about 4 hour and after that i take a reading on UV spectrometer by considering LB as control. Does it show any reading if the number of the bacteria is about 6144 no. ? or it should have limitation... and if extract DNA(impure) does that effect the accuracy and amount of reading?
With kind regards.

[deleted-140482]

Hi Sukhpreet,

I feel like we are sort of combining two separate things here, so I want to make sure that everything is clear. You have been talking about using a spectrophotometer to determine the concentration of DNA and also the concentration of E. coli, but these are not done precisely the same. In the case of DNA, you would take your sample and measure the absorbance using ultraviolet light, at a wavelength of 260nm. You can use the absorbance at 260nm to determine the concentration of your DNA by knowing that 50ug/mL of double stranded DNA (which is what you will have isolated from your cheek cells) gives an absorbance (OD) of 1. So an OD=2 would tell you that you have 100ug/mL DNA. This assumes pure DNA, and you mentioned that you were concerned about concentration. DNA could be contaminated with multiple things that affect absorbance, but the most common one is protein. In order to determine the purity of your DNA, you can measure the absorbance at 260nm and at 280nm. The A260/A280 ratio of pure DNA is ~1.8. Anything lower suggests some protein contamination (which is completely normal and to be expected. It doesn't mean that you still don't have plenty of DNA present). DNA can also be contaminated by RNA, salts, etc. For your purposes, I would guess that you can mostly ignore this, but I'll link to a pretty good article about how each of these can be measured/dealt with. http://www.promega.com/resources/pubhub ... =200715310

Now, you also seem to be concerned about contamination with bacteria. If your original sample has both cheek cells and E. coli in it, and you extract the DNA from both there will be no (easy) way to determine how much is mammalian versus bacterial DNA, but I would expect the amount of contamination from the bacteria to be fairly minimal relative to your cells. Furthermore, bacteria are much harder to lyse than human cells, so you might not be isolating nearly as much DNA from them anyway, depending on your exact protocol. You also seem to be talking about determining the concentration of your E. coli (I assume you mean whole, intact bacteria in this case). We do not use ultraviolet spectrophotometry to determine this, but actually use visible light. Generally, people determine the concentration of bacteria at a wavelength of 600nm (which again, is in the visible light spectrum). In this case you can calculate the concentration by knowing that an OD of 1 (at 600nm) is equal to a concentration of 8x10^8 cells/mL. This is specific to E. coli and will vary somewhat for other types of cells/bacteria. To be honest, I'm not sure what the "minimum" concentration of bacteria is that you would need to be able to read it on a spectrophotometer, but if you are actually growing bacteria in LB broth, you will definitely have enough to read it. However, if you are concerned about the small amounts of intact bacteria you could get from your cheek swab affecting your determination of DNA concentration, I really wouldn't worry. You would have to have enough bacteria present to really affect the turbidity of your solution for that to happen, and I seriously doubt that you'll have that much.

I hope this helps!
JMP