1. Compare and contrast organic, inorganic and solid phase approaches for DNA isolation
Organic isolation takes place after the DNA has been released from the cell and a further purification is to be conducted. The purification is done in order to remove the contaminating proteins, lipids, carbohydrates and cell debris. Organic isolation makes use of a combination of high salt, low pH and an organic mixture of phenol and chloroform. The mixture dissolves hydrophobic contaminants and then strips away most of the DNA associated proteins. The DNA precipitate in this method is collected by centrifugation. The DNA pellet is then dissolved in rehydration buffer. Inorganic isolation method is used in cases that do not require phenol extraction and ensure efficient recovery of clean DNA. Also known as salting out and is similar to the organic isolation as it also uses high salt and low pH. The DNA is precipitated using isopropanol and then suspended in TE buffer or water the same way the organic isolation is done. Solid-phase approach of DNA isolation on the other hand, is more rapid and can be done using solid matrices for washing the DNA. When removing the DNA extracts from the mixture, the bound complementary DNA is eluted by heating the solid matrices or chemically breaking the hydrogen bonds.
2. Compare and contrast organic and solid phase approaches for isolating total RNA
The organic approach for isolating total RNA involves the use of centrifugation to separate reticulocytes in blood and bone marrow. The sample is then frozen in liquid nitrogen to inactivate intracellular RNAses. Chemical lysis is also used for the separation of bacterial and fungal RNA. After the lysis process, the solid are extracted using organic methods which incorporates the use of acid phenol. However, when using the solid state approach, all the above procedures are similar except at the when it comes to the lysis section whereby solid phase uses ethanol to adjust the strong buffering conditions. When using the organic method, Isoamyl alcohol is used to prevent foaming and the phase has to be acidic with a pH value of 4-5. RNAse free DNA is added directly to the isolated RNA which is similar to the case of solid state that also incorporates the addition of DNAse to the adsorbed RNA to remove contaminating DNA. In the solid state method, centrifugation is used to get the resultant RNA whereas organic method uses a 70% ethanol resuspened in RNF buffer to wash the RNA precipitate.
3. Distinguish between isolation of total RNA with that of messenger RNA
In the isolation of total RNA, osmosis is used to lysed the samples which usually consist of reticulocytes in blood and the bone marrow. The components can also be separated from the white blood cells by centrifugation and employs the use of two methods; organic and solid state approaches to isolate total RNA. In isolating the messenger RNA, single-stranded oligomers of thymine or the uracil are immobilized on a matrix resin column or beads. The final RNA product in messenger RNA isolation is enriched in polyA RNA.
4. Describe gel based spectrophotometric and fluorometric methods which are used to determine the quantity and quality of DNA and RNA preparations.
The spectrophotometric approach makes use of the fact that nucleic acid can absorb light at 260nm through adenine residues. It then uses the Beer-Lambert Law to determine the aborbtivity constants using the relationship: