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The following points highlight the four main properties of DNA and RNA. The properties are:- 1. Storage 2. Purification 3. Concentration 4. Fluorescent Quantification of DNA.
Property # 1. Storage:
The following properties of reagents and conditions are important considerations in processing and storing DNA and RNA:
1. Heavy metals promote phosphodiester breakage. EDTA is an excellent heavy metal chelator. Free radicals are formed from chemical breakdown and radiation and they also cause phosphodiester breakage.
2. UV light at 260 nm causes a variety of lesions, including thymine dimers and crosslinks. 320 nm irradiation can also cause crosslinks, but less efficiently.
3. Ethidium bromide causes photo-oxidation of DNA with visible light and molecular oxygen. Oxidation products can cause phosphodiester breakage.
4. Nucleases are found on human skin; therefore, avoid direct or indirect contact between nucleic acids and fingers.
5. Most DNases are not very stable; however, many RNases are very stable and can adsorb to glass or plastic and remain active.
6. 5°C is one of the best and simplest conditions for storing DNA; -20°C is the temperature that causes extensive single and double strand breaks, while -70°C is excellent for long- term storage. For long-term storage of DNA, it is best to store in high salt (>1 M) in the presence of high EDTA (>10 mM) at pH 8.5. Storage of DNA in buoyant CsCl with ethidium bromide in the dark at 5°C is also excellent. There is about one phosphodiester break per 200 kb of DNA per year.
7. Storage of λ DNA in the phage is better than storing the pure DNA.
Property # 2. Purification:
To remove protein from nucleic acid solutions – Treat with proteolytic enzymes, such as pronase, proteinase K.
The simplest method for purifying DNA is to extract with phenol or phenol-chloroform and then chloroform. The phenol denatures proteins and the final extraction with chloroform removes traces of phenol.
Purify by cesium chloride-ethidium bromide density gradient centrifugation.
Property # 3. Concentration:
DNA and RNA solutions are concentrated with ethanol as given below:
The volume of DNA is measured and the monovalent cation concentration is adjusted. The final concentration should be 2-2.5 M for ammonium acetate, 0.3 M for sodium acetate, 0.2 M for sodium chloride and 0.8 M for lithium chloride.
The ion used often depends on the volume of DNA and on the subsequent manipulations; for example, sodium acetate inhibits Klenow, ammonium ions inhibit T4 polynucleotide kinase, and chloride ions inhibit RNA- dependent DNA polymerases.
The addition of MgCl2 to a final concentration of 10 mM assists in the precipitation of small DNA fragments and oligonucleotides. Following addition of the monovalent cations, 2-2.5 volumes of ethanol are added, mixed well, and stored on ice or at -20°C for 20 minutes to 1 hour.
The DNA is recovered by centrifugation in a microfuge for 10 minutes at room temperature. The supernatant is carefully decanted making certain that the DNA pellet, if visible, is not discarded (often the pellet is not visible until it is dry).
To remove salts, the pellet is washed with 0.5-1.0 ml of 70% ethanol, centrifuged again, the supernatant decanted, and the pellet dried. Ammonium acetate is very soluble in ethanol and is effectively removed by a 70% wash. Sodium acetate and sodium chloride are less effectively removed.
For fast drying, the pellet can be centrifuged briefly in a Speedvac, although the method is not recommended for many DNA preparations as DNA that has been over-dried is difficult to re-suspend and also tends to denature small fragments.
Isopropanol is also used to precipitate DNA but it tends to co-precipitate salts and is harder to evaporate since it is less volatile. However, less isopropanol is required than ethanol to precipitate DNA and it is sometimes used when volumes must be kept to a minimum, example, in large-scale plasmid preparations.
Spectrophotometric Determination of Nucleic Acid Purity and Concentration:
Isolated DNA can be stored for a long time in TE buffer. DNA stored in water is subject to acid hydrolysis. Contaminants in the DNA solution will lead to DNA degradation. Avoid repeated freeze-thawing as this will lead to precipitates. Store DNA samples in aliquots.
Spectrophotometry and fluorometry are commonly used to measure microgram quantities of pure DNA samples. Fluorometry is more sensitive, allowing measurement of nanogram quantities of DNA.
The amount of nucleic acid present in a sample can be quantified using the absorbance at 260 nm in a cuvette (quartz) using a spectrophotometer. An Optical Density (OD) of 1.0 is achieved by different concentrations of nucleic acid, depending upon the type of nucleic acids.
OD = 1.0 for 50 μg/ml for double-stranded DNA;
= 40 μg/ml for single-stranded DNA;
= RNA and 20 μg/ml for oligonucleotides (20 mers).
In addition to the absorbance at 260 nm, absorbance at 280 nm is also useful for determining the purity of nucleic acids. The absorbance 260/280 is 1.8 for pure DNA and 2.0 for pure RNA. Deviations from these ratios indicate contaminations of protein and phenol. To determine the concentration of nucleic acid in the sample, the following formula is used.
Total nucleic acid (μg) = [A 260] [OD value (1.0)] [Dilution factor]
For example – if one diluted a double stranded DNA sample 1:99 (= 1 in 100) with pure distilled water and the A260 value from the spectrophotometer was 0.20, the concentration of the DNA would be –
Total DNA (μg) = [0.20] [50] [100]
= 1000 μg/ml = 1 mg/ml as OD = 1.0 for 50 μg/ml
Property # 4. Fluorescent Quantification of DNA:
The amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide in that solution. Dilutions of an unknown DNA in the presence of 2 μg/ml ethidium bromide are compared to dilutions of a known amount of a standard DNA solutions spotted on an agarose gel or Saran Wrap or electrophoresed in an agarose gel.
1. Mix 9 μl TE and 1 μl DNA sample (mix thoroughly or vortex).
2. Spot this on a piece of parafilm, mix by pulling it up and down once, and make a second 10 fold dilution with 9 μl more of TE and 1 μl of the first dilution.
3. Prepare an agarose gel (0.5%-0.7%) with ethidium bromide. Spot 5 μl of these dilutions on different places of the agarose gel (label the bottom of the plate).
4. If the concentration of the original DNA sample is suspected to be less than 20 μg/ml then spot 5 μl of the undiluted sample also.
5. Also spot on the agarose gel 5 μl of at least the 2, 5, 10, 15, and 20 μg/ml DNA standards.
6. Allow the liquid soak in the agarose gel. Invert the plate on a transilluminator and compare. Record the unknown spots for fluorescent intensity to the standard.
7. Use the dilution that gives between 2 and 20 μg/ml reading. Back calculate the amount of DNA by the dilution factor. It is best if the sample spots are coded so that anyone can read them.
Quantification of DNA Using Diphenylamine (DPA) Assay:
1. Prepare the following reagent in a fume hood.
Distilled water – 160 μl
DNA – 10 μl
3N perchloric acid – 150μl
DPA solution – 180μl
2. Cap the tubes and mix by inversion.
3. Incubate in the dark (overnight) at room temperature.
Note the incubation time for future use. A calibration curve will then not be necessary each time.
4. Read the optical density at 600 nm.
Compare with a calibration curve made from measurements with a standardized DNA solution. The curve is only linear between the concentrations of 5-50 μg/ml. Dilution may be necessary for more concentrated solutions.
3 N Perchloric Acid:
Dilute 49.2 ml perchloric acid up to 200 ml with distilled water in a fume hood.
Diphenylamine (DPA) Solution:
In a fume hood, add 20 μl paraldehyde to 198 ml of glacial acetic acid in a 250 ml Erlenmyer flask. Wearing gloves, carefully weigh out 8 g of DPA and add to the above solution, quickly cover with foil, and shake until dissolved. Store in the dark in a fume hood. This solution is stable for 6 weeks.