Isolation and purification of nucleic acids are very important for any molecular biology experiment. It is the starting material for all types of molecular biology experiments like restriction digestion, cloning, PCR, DNA-libraries and sequencing. By manipulating this extracted DNA or RNA, a new DNA with different characteristics can be obtained. Genetic engineering process relies on DNA, RNA and proteins only. The basic process of extracting DNA involves the release of DNA from the cells, purification of the DNA to be used in the experiments. The pH of all solutions is maintained at pH-8.0 throughout the extraction procedure.
The isolation and purification of DNA mainly include four steps:
i. Cell lysis
ii. Enzymatic treatment
iii. Purification of DNA/RNA
iv. Quantification of DNA or RNA
i. Cell lysis
It is needed in order to release the components from the cells. The nature of the treatment will vary widely according to the cell type. For cell lysis we need a culture of bacteria or yeast cells for extracting DNA. The cell walls present in bacteria which are needed to be lysed to release the cell content is carried out by using lysozyme. The other chemicals used are EDTA and SDS detergent. EDTA acts as chelating agent and chelates divalent cations which act as co-factor for enzymes. SDS, an anionic detergent breaks the di-sulphide bonds of the membrane proteins and helps the membrane proteins to degrade. In case of plants, the cell walls are degraded either by enzymatic treatment like use of pectinase, cellulase etc. The alternate method of breaking the cell wall of plants and fungi is by using liquid N2 (liquid Nitrogen) and crushing/homogenizing the cells in mortar-pestle. Further cell lysis is carried out by treatment with mild anionic detergent CTAB (Cetyl Trimethyl Amino Bromine). β-mercaptoethanol can be used to break the thio bonds.
ii. Enzymatic treatment
Removal of RNA from a mixture of nucleic acids is easily achieved by treatment with RNase which is a very heat stable enzyme. Protein contamination can be removed by digestion with a proteolytic enzyme such as proteinase K.
iii. Purification of nucleic acids:
- Phenol chloroform extraction
To get pure DNA solution there is a need to remove proteins and other impurities. This is achieved by extraction with phenol or a mixture of phenol and chloroform. Phenol and chloroform are immiscible with water. When the mixture is vigorously agitated, the proteins will be denatured; lipids and carbohydrates solubilize in the organic solvent and get precipitated at the interphase, when centrifuged at high-speed (12,000 rpm). The aqueous phase containing the soluble DNA form the top layer and the debris forms the bottom phase.
- Alcohol precipitation
After phenol extraction, the aqueous phase which is obtained by above extraction will be free from protein impurities and will be much diluted, so to concentrate the sample alcohol precipitation is done by precipitating nucleic acids using chilled ethanol or isopropanol. A small volume (1/10) of 3M sodium acetate solution is added before precipitation step. The acetate ions help to form the network and thus enhance precipitation. On addition of alcohol, a precipitate will appear which can then be collected at the bottom of the test tube by centrifugation.
As mentioned in above paragraph centrifugation is used in DNA purification for separation of cellular debris from solution and for recovering precipitated nucleic acids. Other method which can be used is cesium chloride or CsCl2 density gradient centrifugation for separation of plasmid DNA from bacterial genomic DNA, or RNA from DNA. Sucrose gradient can also be used for size selection of large DNA fragments when constructing genomic library.
- Plasmid Extraction
American molecular biologist Joshua Lederberg introduced the term ‘plasmid’. Plasmid is an extra- chromosomal DNA of bacterium and can replicate independently. It is small in size, double stranded and circular in shape. Due to its self-replicating property; it is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Plasmid size ranges from 1-1000 kilo base pairs.
Plasmids are transferrable genetic elements and also known as ‘replicons’, which are naked DNA and are important tools in labs where they are commonly used to multiply or express particular genes.
Alkaline lysis is a method, to isolate plasmid DNA in molecular biology, in which bacterial cells with the desired plasmid are lysed under alkaline conditions. Bacteria having the plasmid DNA (of interest) is first grown and then allowed to lyse with an alkaline lysis buffer. Lysis buffer consists of a detergent sodium dodecyl sulfate (SDS) and a strong base NaOH (sodium hydroxide). The detergent cleaves the membrane phospholipid bilayer and the alkali helps to denature the proteins that are involved in maintaining the cell membrane structure. Through a series of steps involving agitation, precipitation, centrifugation and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.
Proteins are contaminating agents in any type of DNA isolation so as in plasmid DNA isolation also. They can interfere with the final product and result with low yield. SDS is used to denature the proteins and facilitate the DNA purification process. Agarose gel electrophoresis is a powerful separation method frequently used to analyze plasmid DNA.
The different forms of the same plasmid DNA molecule have the following rates of migration (in decreasing order):
Super coiled > linear > nicked circles > dimer > trimer > etc.
- Column purification
Mainly two types of chromatography can be done for purification of nucleic acids:
- Size exclusion chromatography
- Affinity chromatography.
In size exclusion chromatography, sample mix is passed through a matrix of small porous beads. Smaller molecules such as salts and some nucleotides will enter the beads whereas larger molecules such as longer nucleic acid will pass right through the column.
In affinity chromatography, the macromolecule will bind to resins of column. The resins could be an anionic resins or oligo-dT sequences which specifically bind to the poly-A tail of eukaryotic mRNA molecules. In both chromatography cases undesirable molecules can be washed from the column and elution of nucleic acid bound to column can be eluted out by changing pH conditions or other.
iv. Quantitation by spectrophotometer: The most common technique to determine DNA yield and purity is measurement of absorbance. Pure preparations of DNA and RNA have OD260/OD280 values of 1.8 to 2.0, respectively. If this ratio has value greater than 1.8 for DNA sample then there will be RNA contaminations in the sample. If there is contamination due to protein or phenol, the ratio decreases as compared to the above values and it becomes impossible to find accurate values. Spectrophotometer can be used to measure amount of UV irradiation absorbed by the bases, if the sample is pure. (UV spectroscopy is the most sensitive labile free quantification method for proteins).