Genomic DNA comprises of all the genes that are found in a living organism. This DNA can be extracted to screen for genes of interest. Genes are transcribed to mRNA which is in turn translated to proteins as is necessary. This most of the times is referred to as gene expression as the phenotype that the gene codes for is seen only after it is in a protein form, to give the character that is required. In biotechnology, during genetic engineering, different genes are cloned into a plant or any other organism to give particular characteristics as is desired by the genetic engineer. The researcher then has to be able to determine that the gene has been successfully cloned into the genomic DNA of the said organism. This then calls for screening of the gene and then in most cases even to determine whether the gene is being expressed. Therefore DNA has to be extracted and also RNA to do these molecular analysis.
The CTAB Method
• The cheapest, successful and high yielding way to extract DNA and RNA from plant tissue is using the CTAB (Cetyltrimethylammonium bromide) method. CTAB buffer components are
• 2% CTAB- CTAB is a detergent and it disrupts plant cell walls and biological membranes at the same time denaturing or inhibiting proteins (enzymes).
• 2% PVP-PVP (Polyvinylpyrrolidone-40) precipitates polyphenolics, removing them from the extract.
• 100 mM Tris-HCl (pH8). Tris (hydroxymethyl) aminomethane hydrochloride) gives the right pH for nucleic acid extraction and also helps to makes the cell membranes more permeable.
• 25 mM EDTA- EDTA (Ethylenediaminetetraacetic acid) is a chelating agent that chelates divalent metal ions and prevents the magnesium-mediated aggregation of nucleic acids with proteins and with each other.
• 2 M NaCl- NaCl (sodium chloride) provides the positive ions (Na+), to neutralise the negative charges in the nucleic acids thus bringing the nucleic acid molecules together.
• 0.05% Spermidine. Spermidine binds to and precipitates nucleic acids.
• Autoclave the above after adding distilled water to the required buffer volume.
• 2-3% Î²-mercaptoethanol (added directly before use) - An antioxidant/reducing agent that breaks down intramolecular protein disulfide bonds thus improving denaturing of proteins.
1. Homogenise ~250 mg of the plant tissue using pestle and mortar in liquid nitrogen and transfer into a 2 ml eppendorf.
2. Add 1 ml of pre-warmed (at 65 Â°C) CTAB buffer and vortex for 30 -60 seconds
3. Incubate for at least 30 minutes at 65 Â°C .
4. Centrifuge for 10-20 minutes at room temperature at 16000 xg.
5. Transfer supernatant to a new 2 ml eppendorf and add equal volume of 24:1 chloroform: isoamyl alcohol and centrifuge at 13 000 xg for 2-5 minutes.
Chloroform denatures proteins and causes them to precipitate and the foaming of proteins that is normally caused by the extraction mechanism is reduced by the isoamyl alcohol.
6. Repeat above.
7. Add 600 Âµl ice cold isopropanol ( precipitates the DNA and removes salts that are alcohol-soluble) and incubate at -20 Â°C for 10 minutes.
8. Centrifuge at 13 000 xg for 2-5 minutes.
9. Remove supernatant and add 1ml of 70 % ethanol( to wash the DNA) and allow to stand for 30 minutes.
10. Centrifuge at 13 000 xg for 2 minutes.
11. Remove the ethanol and allow to dry ( can be dried overnight or as long as it can take, using a speed vac is the fastest option).
12. Then dissolve in 50-100 Âµl TE buffer ( comprised of Tris-HCl (pH8) and EDTA).
For RNA extraction the above steps from steps 1-6 are followed but now centrifuging at 4 Â°C for 15 minutes at steps 5 and 6. This is then followed by
7. Adding 1/3 volume of 8 M LiCl (Lithium Chloride) and incubate for 2 hours at 4 Â°C (preferably overnight) to precipitates the RNA. LiCl does not precipitate DNA, proteins and carbohydrates efficiently, but does so efficiently for RNA.
8. Centrifuge for 60 minutes at 4 Â°C at 16 000 xg
9. Remove the supernatant and wash with 800 Âµl-1000 Âµl 70 % ethanol and centrifuge for5-15 minutes
10. Remove ethanol and allow to dry.
11. Then resuspend the pellet in 30 Âµl-50 Âµl RNase free double distilled water.
Quantifying and Assessing the Quality of the DNA and RNA
Ultraviolet radiation spectroscopy can then be used to get the concentration of the DNA and RNA isolated from the plant tissue. This is measured at 260 nm and 280 nm. This method cannot distinguish between DNA and RNA which is why the RNA sample has to be RNase free so that the RNA is not degraded and then DNase has to be added to the RNA sample to degrade any contaminating DNA. The concentration is then calculated using the Beer-Lambert law A=ECI.
The ratio of 1.8-2.1 of A260/A280 indicates a highly pure RNA sample and the 260/230 ration has to be greater than 2 but less than 2.4. A nanodrop offers an easier and faster way to quantify the DNA and RNA giving the ratios to determine purity at the same time.
RNA quality can be assessed using native 1% agarose gel electrophoresis. To do this an RNA loading buffer (about 3 Âµl of a 10X loading buffer) is added to 1Âµg of RNA and incubated for 5-10 minutes at 65 Â°C, snap frozen and electrophoresed. It is then visualised under UV light and 2 distinct bands representing the 28S and 18S rRNA can be clearly and sharply be visualised with the 28S being brighter that the 18S bands.
To check for the gene of interest in the extracted genomic DNA , polymerase chain reaction (PCR) gene specific primers are to amplify the gene since the its size is known, the PCR product is then electrophoresed on 1 % agarose gel stained with ethidium bromide and viewed under UV light. To check for gene expression, the mRNA is reverse transcribed to get cDNA, after which Northern blotting techniques or RT-PCR can be used to see if the gene is being expressed.
After doing this then the researcher is confident that the gene of interest is functional. Its activity can be characterised by comparing the untransformed plant to the transformed one, thus determining the effect of the gene in the plant. Then other analytical techniques can be carried out such as phytochemical analysis, microarrays etc.
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