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The following points highlight the top three methods used for estimation of DNA. The methods are: 1. Witham et. al.( 1971) 2. Ethidium Bromide Method 3. U V Spectrophotometric Method.
1. Method by Witham et. al.( 1971):
Principle:
DNA is initially depurinated quantitatively, followed by the dehydration of sugar to hydroxylevulinylaldehyde. In an acidic medium, this aldehyde condenses with dipheny-lamine to produce a condensation product which is deep blue with an absorption maximum at 595 nm.
Requirements:
Reagents:
1. Diphenylamine (DPA)-Dissolve 1.0 g DPA in 85 ml glacial acetic acid. Add 2.75 ml concentrated H2SO4 and make up the volume to 100 ml with glacial acetic acid. This is stable for 6 months at 2°C. Before use, bring to room temperature and swirl.
2. Standard DNA-Dissolve 100 mg in 100 ml distilled water (1 mg/ml).
3. Sample DNA.
4. Graduated pipettes.
5. Tubes and stands.
6. Spectronic 20.
7. Glass marking pencil.
8. Water bath.
Procedure:
1. Prepare separate tubes with 0.5, 1.0, 1.5,2.0 and 2.5 ml of standard DNA and 0.5 ml and 1.0 ml of sample DNA along with 2.5 ml distilled water as blank.
2. Make up the volume in all tubes to 2.5 ml and add 4 ml of diphenyl amine reagent. Heat the tubes to boiling on a water bath for 10 minutes and cool.
3. The blue colour developed is read at 595 nm.
4. Plot a graph and calculate amount of DNA in sample.
2. Ethidium Bromide Method:
For the estimation of nucleic acids there are several methods available. One of these is the ethidium bromide method. Here nucleic acids absorb U.V. light at 260 and 280 nm and bind the fluorescent dye ethidium bromide. This method is useful when small amounts of DNA or contaminating U.V. absorbing material are present.
However, ethidium bromide is a carcinogen and hence should be handled and disposed carefully. Hoescht dye 33258 is an alternative to ethidium bromide but it requires the use of a fluorimeter, which is expensive.
Spectrophotometric method is useful when sufficient quantities of relatively pure DNA are to be estimated.
Principle:
Accurate measurement of DNA or RNA can be done by absorbance at 260 nm with small or dilute quantities and requires only µg or ng amount of sample.
Requirements:
Reagents:
1. DNA standard 0.1 µg/ml:
Dissolve lambda DNA in TE to ~ µg/ ml. Then determine the concentration accurately by A260 (an A260 of 1 = 50 µg. DNA/ml or 40 µg RNA/ml).
Dilute to 100 ng/ml in TE and prepare dilution of 90, 80, 70, 60, 50, 40, 30, 20 and 10 ng/ml from this stock.
2. DNA samples.
3. Ethidium bromide stock solution 10 mg/ ml.
4. UV transilluminator with poloroid camera/image analyser.
5. Petri dishes.
6. Glass plate.
7. Micropipettes, tips and tubes.
8. Goggles for UV blocking.
9. Waste container for ethidium bromide disposal.
Procedure:
1. On Petri dish or glass slide spot 10 µl aliquots of 10,20,30,40,60, 80 and 100 ng/ml lambda DNA (or ribosomal DNA depending on which sample you are using).
2. Prepare 1:2 dilutions of the sample DNA/ RNA (usually 4-8 are enough) in TE.
3. Spot 10 µl of each of these dilutions on to Petri plate.
4. Add 10 µl of 0.2 µg/ml ethidium bromide to each spot and mix well (by pipetting up and down).
5. Using transilluminator/ a hand held U V source, photograph the plate.
6. Estimate concentration of sample by comparing dilutions of samples with each of the standard DNA/RNA.
3. U V Spectrophotometric Method:
Requirements:
1. DNA samples.
2. SSC buffer 0.1x.
3. UV spectrophotometer.
4. Quartz cuvettes.
Procedure:
1. Dissolve small quantity of extracted DNA in 3.0 ml of 0.1 x SSC buffer.
2. Using 0.1 x SSC buffer as blank determine the absorbance of DNA samples at 220 on UV spectrophotometer.
3. Repeat with same blank and samples of DNA at 230 nm.
4. Repeat with 10 nm increase in wave length with blank and sample till 300 nm and record absorbance.
5. The ratio of 260 to 280 nm absorbance is to be computed. Pure DNA (without RNA or protein) will have a 260:280 absorbance ratio of 1.85. For RNA, 260: 280 ratio will be 2.0.
6. Plot absorbance spectrum of the sample and indicate 260:280 nm ratio and also the protein contamination on the graph.