Nucleic Acid

The deoxypentose present in DNA as deoxyribose give this reaction. In the presence of acidic medium, the DNA gets depurinated and dehydrated to form ω-hydroxylevulinylaldehyde. This aldehyde then reacts with diphenylamine to produce a deep blue colored product, which can be easily quantified. The pentose sugar present in RNA then reacts with concentrated hydrochloric acid in a boiling water bath to form furfural. The furfural then reacts with orcinol to give a green colored compound. Ferric chloride acts as a catalyst. Only purine nucleotides give a significant reading. The plasmid DNA is extrachromosomal, covalently closed circular DNA in bacteria. It can be obtained from bacterial suspension when it is subjected to a high pH and strong anionic detergent. Under this condition, the bacterial cell wall opens up, the DNA and proteins get denatured and on shifting the pH to neutral, the strands of plasmid DNA get renatured while the genomic DNA cannot get renatured. The DNA is precipitated using ethanol and is collected by centrifugation. The boiling of bacterial suspension in the presence of lysozyme and TritonX-100 is used to obtain the plasmid DNA. Lysozyme attacks the cell wall of bacteria while TritonX-100 disrupt the lipid-lipid and lipid-protein interaction. The heat cracks open the bacterial outer shell and also disrupt the hydrogen bond between the bases of DNA. Heat denatures the proteins and DNA; the plasmid DNA again renatures when the temperature is lowered. The denatured chromosomal DNA and proteins get separated by centrifugation. The protocol is used to purify plasmid DNA. It involves the treatment of a crude mixture of nucleic acid with lithium chloride. The treatment of lithium chloride precipitates large RNA and proteins. This is followed by treatment with RNase. The plasmid DNA is purified by extraction with phenol-chloroform followed by ethanol precipitation. The genomic DNA of an unknown bacteria strain can be obtained by the method involving enzymes for degradation of the cell wall of both Gram-positive and Gram-negative bacteria. It involves achromopeptidase, which has bacteriolytic activity against the cell wall of Gram-positive bacteria, as well as lysozyme, which has bacteriolytic activity against Gram-negative bacteria. The protocol involves SDS, to solubilize the lipids and proteins of the cell membrane and proteinase K, which acts on the proteins and digests them. This is followed by phenol-chloroform extraction and ethanol precipitation. The process of isolation of DNA from plants involves the inclusion of CTAB, which aids in the separation of polysaccharides. It also includes reagents such as polyvinylpyrrolidone which help in the removal of polyphenols. Both polysaccharides and polyphenols form a significant proportion of plant materials. This is followed by the extraction of DNA with phenol-chloroform and precipitation with ethanol. The DNA can be obtained from eukaryotic cells by the addition of proteinase K which denatures the proteins. Proteins get precipitated using ammonium acetate. This is followed by the extraction of DNA with phenol-chloroform and precipitation with ethanol. The DNA can be obtained from eukaryotic cells by the addition of proteinase K which denature the proteins. Proteins get precipitated using ammonium acetate. This is followed by the extraction of DNA with phenol-chloroform and precipitation with ethanol. The process of agarose gel electrophoresis of DNA is a method used to separate and identify the DNA fragments. Agarose is a linear polymer, it comprises alternate d- and l-galactose joined by α(1-3) and β(1-4) bonds with anhydro bridge between 3 and 6 positions. It is gelatinized by heating it with buffer and casted in a tray with combs in it. Ethidium bromide can be incorporated in the gel or the gel can be stained with it after electrophoresis. On solidification the combs are removed to form wells. The DNA sample is filled in the wells with a loading dye and the tank is filled with buffer. The electrophoresis is allowed to take place after connecting it with a power supply. After electrophoresis, the gel is observed under a transilluminator. This method is used to study the organization of genes in the genome. This technique combines electrophoresis and hybridization. In this, DNA is digested with restriction enzymes and the resulting fragments are subjected to electrophoresis in an agarose gel which separates them according to their size.Separation of DNA fragments is followed by its in-situ denaturation and transfer of gel to a membrane. The membrane with the DNA is then hybridized to a labeled oligonucleotide probe. The binding of the DNA to its probe is detected by a detection system, e.g., autoradiography.After comparing the bands generated by the digestion of genomic DNA with restriction enzymes, it is feasible to place a target DNA in a genome. Northern hybridization is used to measure the size, abundance, and amount of RNAs. In this, the RNA is isolated from the cells and then separated in a denaturing agarose gel. Separation of RNA fragments is followed by its in-situ denaturation and transfer of gel to a membrane. The membrane with the RNA is then hybridized to a labeled oligonucleotide probe. The binding of the RNA to its probe is detected by a detection system, e.g., autoradiography.