Read this article to learn about the isolation and separation, electrophoresis and blotting method of nucleic acid.
Contents
Isolation and Separation of Nucleic Acids:
Isolation of DNA:
DNA is recovered from cells by the gentlest possible method of cell rupture to prevent DNA from fragmenting by mechanical shearing.
This is usually in the presence of EDTA which chelate the Mg2+ needed for enzymes that degrade DNA, termed deoxyribonucleases (DNases). Ideally, cell walls, if present, should be digested enzymatically (e.g., lysozyme treatment of bacteria), and the cell membrane should be solubilized using detergent.
If physical disruption is necessary, it should be kept to a minimum and should involve cutting and squashing of cells, rather than the use of shear forces. Cell disruption and most subsequent steps should be performed at 4°C using glassware and solutions that have been autoclaved to destroy DNase activity.
After release of nucleic acid from cell, RNA can be removed by treatment with ribonucleases (RNase) that have been heat treated to remove any DNase contaminants as RNase is stable to heat because of its di-sulphite bonds, which ensure rapid renaturation of molecule on cooling. The other major contaminant, protein, is removed by shaking the solution gently with water saturated phenol, or with a phenol/chloroform mixture, subsequently after proteinase K treatment, either of which denatures proteins, not nucleic acids.
Centrifugation of the emulsion formed by this mixing, produces a lower organic phase and upper aqueous phase separated by interface of denatured proteins. The aqueous phase is recovered and deproteinised repeatedly until no more material is seen in interface.
Finally the deproteinised DNA preparation is mixed with two volumes of absolute alcohol, and DNA pellet is re-dissolved in a buffer containing EDTA to inactivate any DNase present. The solution can be stored for at least a month at 4°C. DNA can also be stored frozen but repeated freezing and thawing tends to damage long DNA molecules by shearing.
If DNA from cell organelle or viral particle is needed, it is better to isolate the cell organelle or viral particle first as recovery of DNA from a mixture of DNA is difficult. Where high degree of purity is required, DNA may be subjected to density gradient ultra-centrifugation through caesium chloride, which is particularly useful for the preparation of plasmid DNA. It is possible to check the integrity of DNA by agarose gel electrophoresis and determine the concentration of DNA by using the fact that 1 absorbance unit equates to 50 µg DNA cm-3 and so,
50 × A260 = concentration of DNA sample (µg cm-3)
Contaminants may also be identified by scanning ultraviolet spectrophotometry from 200- 300 nm. A ratio of absorbance at 260 nm to that at 280 nm of approximately 1.8 indicates that the sample is free from protein contamination which absorbs strongly at 280 nm.
Isolation of RNA:
RNA molecules are relatively short and thus are less prone to damage by shearing, so can be subjected to vigorous cell disruption. RNA is very vulnerable to digestion by RNase which are present endogenously as well as exogenously on finger tips. Gloves should, therefore, be worn and strong detergent should be included in the isolation medium to immediately denature any RNase.
Subsequent deproteinisation is done vigorously as RNA is usually tightly associated with protein. DNase treatment is then followed to remove DNA contamination and at last RNA is precipitated with ethanol. Commonly used treatment in RNA extraction is guanadinium thiocyanate, which is both strong inhibitor of RNase and strong denaturant.
The integrity of RNA extracted can be checked by agarose gel electrophoresis. The most abundant RNA species are rRNA molecules 23s and 16s in prokaryotes and 18s and 28s in eukaryotes. The concentration of RNA may be estimated by using ultraviolet spectrophotometry. At 260 nm 1 absorbance unit equates 40 µg cm-3 of RNA and therefore:
40 × A260- concentration of RNA sample (µg cm-3)
Contaminants may also be identified in the same way as that for DNA by scanning ultra violet spectroscopy; however, in the case of RNA a 260 nm: 280 nm ratio of approximately 2 would be expected for a sample containing no protein.
In many cases it is desirable to isolate eukaryotic mRNA, which is only 2-5% of total RNA molecules. At high salt concentrations, the mRNA containing poly (A) tails binds to the complimentary oligo (dT) molecules of the affinity column so mRNA will be retained and all other RNA molecules will be washed off by further high salt solutions.
Finally, bound mRNA can be eluted by using low concentrations of salt. Nucleic acid species may also be sub-fractionated by more physical means like electrophoresis and chromatographic separation based on the differences of nucleic acid fragment sizes or physicochemical characteristics.
Electrophoresis of Nucleic Acids:
Electrophoresis in agarose or polyacrylamide gel is the usual way to separate DNA molecules according to the size. The technique can be used analytically or preparatively, and can be qualitative or quantitative. Large fragments of DNA such as chromosomes can be separated by a modification of electrophoresis termed as pulsed-field gel electrophoresis (PFGE).
The easiest and common method is electrophoresis in horizontal agarose gels, followed by staining with ethidium bromide. This dye binds to DNA by insertion between stacked base pairs (intercalation), and exhibits a strong orange/red fluorescence when illuminated with ultraviolet light.
Very often electrophoresis is used to check the purity or intactness of a DNA preparation or to assess the enzymatic activity during cloning process. Agarose gels are used to separate DNA molecules larger than 100 bps while shorter DNA fragments can be separated easily with polyacrylamide gels.
When electropohresis is done preparatively, DNA is recovered from gel by cutting the DNA containing fragment of gel with scalpel followed by different treatments like crushing with glass rod in presence of agarase to digest agarose and setting DNA free or by process of electro- elution in which the gel sealed in a dialysis tubing is placed between two electrodes in presence of buffer. Passage of an electrical current between electrodes causes DNA to migrate out of the gel piece but it remains trapped within the dialysis tubing and can, therefore, be recovered easily.
Nucleic Acid Blotting Method:
Electrophoresis allows separation on the basis of size but it does not allow the detection of specific fragment among the complex sample. This can be achieved by transferring the DNA from gel onto nylon or nitro cellulose membrane thus providing a more permanent record of the sample. The method is called southern blotting.
In this, first the gel is soaked in alkali to render the DNA single stranded. It is then transferred to the membrane in the pattern exactly similar in the gel. This transfer can be brought electrophoretically or by drawing large volume of buffer through both gel and membrane, thus transferring DNA by capillary action.
Now the transferred DNA molecule can be probed with labelled DNA molecule called gene probe. This single stranded gene probe will hybridize under right conditions to complimentary fragments immobilized onto the membrane. For hybridisation to take place effectively proper concentrations of salts and adequate temperature are very necessary.
This is usually referred to as stringency of the hybridisation and is particular for each gene probe and sample of DNA. Then unbound probe is removed by washing with buffer and membrane is developed like photographic film after which the precise location of the probe and target can be visualized.
The same basic process of nucleic acid blotting can be used to transfer RNA from gels onto similar membrane. This allows identification of specific mRNA sequences of definite length by hybridisation to a labelled gene probe and is known as “Northern blotting”. By this one cannot only detect but also can quantify the relative amount of specific mRNA. For this mRNA transcripts are separated by electrophoresis under denaturing conditions which improve resolution and also aids in more accurate estimation of the sizes of transcripts.
The format of the blotting may be altered from transfer from gel to direct application to slots on a specific blotting apparatus containing the nylon membrane. This is termed slot or dot blotting and provides a convenient means of measuring the abundance of specific mRNA transcripts without the need for gel electrophoresis; however, it does not provide information regarding the size of fragments. 