Nucleic Acids (Lab) Flashcards
Repository of genetic information and blueprint for all organism
Nucleic Acids
basic structural forms (2):
Deoxyribonucleic acid (DNA)
➢Genetic material
➢Provides template for RNA transcription
Ribonucleic acid (RNA)
➢Carriers of genetic information for protein translation
Factors to consider in DNA (& RNA) Isolation (6):
➢High content of DNA (& RNA)
➢Nucleic acid – major component of all cells (5-15% of cell’s dry weight)
➢Low deoxyribonuclease (DNase) and ribonuclease (RNase) activity
➢Nucleic acids isolated from source can be obtained in pure form.
➢Large size and fragile nature of DNA (Difficulty in isolating intact and undamaged DNA; High-molecular weight and intact DNA vs. sheared or fragmented DNA)
➢Compared to DNA, RNA is less stable (Single-stranded molecule; Presence of 2’-hydroxyl group)
Experimental (Physical & Chemical) Factors (6):
➢pH: extremes in pH
➢Temperature: heating
➢Ionic strength
➢Cellular conditions
➢Mechanical stress
➢Storage of DNA/RNA
Samples for DNA Isolation (4):
➢Tissues containing cells with (↑ nuclear-cytoplasmic volume ratio)
➢DNA concentration is high
in the nucleus.
➢Organs rich in nucleic acids (Spleen, liver, thymus and pancreas versus brain and muscle tissues – lower concentration)
➢Thymus (the best source of DNA.)
Interactions found in nucleic acids (3):
➢Phosphodiester bonds
➢Hydrogen bonds (involved in the complementary base pairing in DNA)
➢Van der Waal’s forces (pi-pi
complexation; Base stacking – stacking
interactions)
General Steps in DNA Isolation (3):
- Homogenization – lysis or disruption of
biological membranes
- Mechanical – mincing, grinding, sonication and etc.
- Chemical – detergents & chaotropic agents - Dissociation and denaturation of the
nucleoprotein
- Deproteinization - Purification of nucleic acid
General Steps in DNA Isolation (3):
- Homogenization – lysis or disruption of
biological membranes
- Mechanical – mincing, grinding, sonication and etc.
- Chemical – detergents & chaotropic agents - Dissociation and denaturation of the
nucleoprotein
- Deproteinization - Purification of nucleic acid
Isolation of DNA from Onion (A. cepa):
- The homogenizing solution contains:
➢0.15 M NaCl [precipitates nucleoproteins (salting-out)]
➢5% SDS (sodium dodecyl sulfate) (breaks ionic interaction between protein and nucleic acid)
➢0.15 M sodium citrate [Chelate Ca2+ and Mg2+ ions (cofactors)]
➢1 mM EDTA [Chelate Ca2+ and Mg2+ ions (cofactors)]
Isolation of DNA from Onion (A. cepa):
➢Heating at 60°C
- dissolves nucleic acids
➢Cooling in ice bath
- retards nuclease activity
➢Papain from meat tenderizer or 6% bromelain
- denatures protein
➢Ice-cold 95% EtOH
- precipitates DNA and RNA
➢Ice-cold 100% iPrOH
- Precipitates DNA only
➢Alcohol (EtOH) and ether (organic solvents)
- Removes lipids
{preserves the integrity of DNA by maintaining the pH of the solution}
➢TE Buffer
➢SSC (Standard Saline Citrate) Solution
Hydrolysis of Nucleic Acids (2):
Acid Hydrolysis
Alkaline (basic) Hydrolysis
Not complete for RNA (Mixture of 2’ and 3’-
nucleotides)
Alkaline (basic) Hydrolysis
not readily hydrolyzed by dilute alkali
DNA
Chemical Characterization of Nucleic Acids
Test for Phosphates’ positive result:
Yellow Crystalline Precipitate
Test for deoxyribose: diphenylamine or Dische (1930) test:
Reagent: diphenylamine in conc. H2SO4
Positive Result: blue complex/compound
