BIOCHEM LAB LAST NA BUTI NAMMAN

• The genetic information of all living organisms is contained within the sequence of the nucleic acids
• Ribonucleic acid (RNA) contains the sugar ribose
• deoxyribonucleic acid (DNA) contains deoxyribose
• the genetic information is contained in DNA
• in certain viruses, this information is contained in the RNA.
• Among the bases, uracil is exclusively found in RNAs.
• The complete set of nucleic acids containing the genes for an organism is referred to as the genome
• Chromosomes are thread-like structures located inside the nucleus of living cells.
• chromosome is made of protein and a single molecule of deoxyribonucleic acid (DNA).
•  In prokaryotes (Greek pro, “before,” and karyon, “nucleus”)
• chromatin (a collective term for the DNA and its associated proteins and macromolecules) occurs free-floating in cytoplasm, as these cells lack organelles and a defined nucleus.
• eukaryotes (Greek eu, “new”), the DNA is enclosed in a membrane-bound nucleus.
• (Isolation of Nucleic Acids)amplified fragment length polymorphism (AFLP) analysis
• (Isolation of Nucleic Acids) Random amplified polymorphic DNA (RAPD) analysis
• Recombinant DNA technology involves the biochemical manipulation of genes by using enzymes that normally participate in DNA or RNA metabolism (e.g., restriction enzymes, ligases, etc.). These enzymes are isolated from a variety of organisms and can be commercially obtained.
•  isolation of purified DNA from plants is challenging because of secondary metabolites and other products that interfere with the isolation process, tend to copurify with DNA and interact irreversibly with proteins and nucleic acids.
• Most methods of nucleic acid extraction are based on the use of fresh or frozen tissue, although techniques involving lyophilized tissues are reported to be more advantageous.
• Many protocols of nucleic acid isolation from plant tissues involve the use of acid, alkali, detergents
• detergents (e.g., sodium dodecyl sulfate, SDS, or hexadecyltrimethyl-ammonium bromide, CTAB),
• suitable buffer (e.g., tris(hydroxymethyl)aminomethane, Tris), neutral salt, or various combinations of these extractants.
• EDTA is often included in the extraction buffer to chelate magnesium ions, a necessary co-factor for nucleases, for this purpose.
• The initial DNA extracts often contain a large amount of RNA, proteins, polysaccharides, tannins and pigments, which may interfere with the extracted DNA and difficult to separate.
• Most proteins are removed by denaturation and precipitation from the extract using chloroform and/or phenol.
• Chloroform is often used with phenol since it is also a protein denaturant, and it also stabilizes the rather unstable boundary between the aqueous phase and the pure phenol layer.
• SDS also inhibits nucleases and helps separate the proteins from the nucleic acids, especially in ribosomes
•  RNAs, on the other hand, are normally removed by treatment of the extract with heat-treated Rnase A.
• Polysaccharide and similar contaminants are removed by treatment with NaCl solutions whose concentration is between 0.7 M and 6 M, in combination with CTAB.
• roteinase K is also used to digest proteins that may interfere with the extraction.
• Agarose gel electrophoresis is a separation technique which is based on the molecular weight and size of the DNA.
• Staining is usually done with ethidium bromide to visualize DNA fragments.
• ethidium bromide is a flat planar molecule, it is able to intercalate between the stacked base pairs of DNA.
• This binding results in distortion of the double-helical structure and localized unwinding of the helix. Ethidium bromide will bind very efficiently to double-stranded DNA, but less so to single-stranded DNA and RNA because of the relative lack of base stacking.
• Measurement of absorbance at 260 nm is a well-established method for estimation of concentration of nucleic acids.
• The resonance structures of pyrimidine and purine bases are responsible for the absorption maxima at 260 nm
• physical properties of the solution as the ionic strength and pH can shift the absorption maxima to lower wavelengths
• When the absorptivity is known, the concentration of a certain solution can be determined from the absorbance measurement using the Beer-Lambert law.
• The Beer Lambert Law states that absorbance is a direct function of the thickness b and concentration c of the solution containing the absorbing chemical species and the absorptivity a of the species, which is characteristic of the species at a given wavelength
• The absorbances of species at a certain wavelength are cumulative; hence, the presence of interferent species (e.g. proteins in nucleic acid determination or nucleic acids in protein determination) can alter the accuracy of quantitation.
•  measurement at more than one wavelength, e.g. 260 and 280 nm, from which a ratio can be determined, is a check of the validity of the A260.
• reading for nucleic acid quantitation. It is also a means of estimating whether the nucleic acid is contaminated with protein or other materials