Experiment 4
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Transcript of Experiment 4
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EXPERIMENT 4NUCLEIC ACIDS
I. NUCLEIC ACIDSFunction: storage and transmission of genes2 types: DNA & RNA3 Components:
Nitrogenous Bases (nucleobase) Sugars (ribose or deoxyribose) Phosphates
Solubility of Nucleic Acids
1. DNA is soluble in H2O2. Effects of salts on DNA in solution:
Salts (eg. Na+ and Mg+2) stabilizes DNA ionic conc. = solubility of DNA; protein solubility ionic conc. = solubility of DNA; protein solubility
3. Effects of organic solvents on DNA:Organic solvents solubilize the hydrophobic core of DNA (destabilizing H-bonds)
4. Effects of pH on DNA/RNAHigh pH (11-12) = ssDNA is stable; RNA is degradedLow pH = DNA bases are removed (apyrimidic or apurinic sites) bec glycosidic bonds are cleaved.
II. ISOLATIONGeneral steps involve in isolation & purification of DNA:1. Disruption of cells & membrane-bound structures to release DNA (homogenization)2. Inactivation of enzymes that hydrolyze the DNA3. Dissociation & denaturation of proteins4. Solvent extraction & concentration of the DNA by precipitation
Reagents used Purpose
1. Homogenizing solution:
a. SDS (sodium dodecyl sulphate)
emulsifies cell lipids/proteins causing cell membrane to breakdown; disrupts polar interaction that hold the cell membrane together
b. EDTA (ethylenediamminetetraacetic acid)
Chelates Ca2+ and Mg2+ cofactors leading to inactivation of DNase
c. NaCl provides isotonic environment dissolves deoxyribonucleoprotein stabilizes the DNA
d. Sodium citrate buffer
2. Meat tenderizer Hydrolyses proteins that can contaminate DNA
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(contains bromelain)
3. Ice cold 95% ethanol Prepitates nucleic acidsThe cold environment decreases the solubility of nucleic acids facilitating faster precipitation.
III. HYDOLYSIS OF NUCLEIC ACIDSA. ACID HYDROLYSIS
Disrupts: (1) H bonding between complementary bases (2) 3’,5’ phosphodiester bond between nucleotides (3) phosphoester bond between phosphate group and sugar component of the
nucleotide (4) N-C glycosidic bonds between sugar component and nitrogenous base of the
nucleoside1. With dilute acids (0.1 N TCA, HCl, or HClO4)
-nucleic acids will ppt. out -RNA boiled in dilute acid (1N HCl, 100C, 1 hr) – liberates A & G leaving “apurinic acid”
2. With stronger acids at higher T (1N TCA, HCl, HClO4; 100C, 15 mins.) purine bases of DNA are cleaved from 2-deoxyribose = depurination
B. BASE HYDROLYSIS - performed on RNA - 100C w/ 0.3 M NaOH
- phosphodiester linkages of RNA are cleaved forming 2’ & 3’-phosphoribonucleotides or 2’,3’-cyclic monophosphonucleotides - DNA not readily hydrolysed by dilute base because it has no 2’ hydroxyl group and will not form 2’,3’-cyclic monophosphonucleotides
IV. UV MEASUREMENT OF NUCLEIC ACIDS-determines the amount and quality of nucleic acids present in solution- 1.6 – 1.8 pure DNA 1.9 – 2.0 pure RNA
Wavelength Absorbing species
230 Carbohydrates
260 DNA and RNA
280 Proteins
Low A260/A280 DNA/RNA is contaminated with proteins
Low A260/A230 DNA/RNA is contaminated with carbohydrates
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V. QUALITATIVE TESTSA. BIAL’S TEST (TEST FOR RIBOSE)
REAGENTS: Orcinol, FeCl3, HCl
(+) VISIBLE RESULT: bluish-green solution
PRINCIPLE INVOLVED:
dehydration forming furfural & condensation with orcinol
TEST FOR: Pentoses
B. DISCHE TEST or DIPHENYLAMINE TEST (TEST FOR DEOXYRIBOSE)REAGENTS: Diphenylamine, glacial acetic acid, concentrated H2SO4
(+) VISIBLE RESULT: Blue solution
PRINCIPLE INVOLVED:
1) dehydration of deoxyribose forming -hydroxylevulinaldehyde2) complexation reaction w/ diphenylamine
TEST FOR: DEOXYRIBOSE
C. WHEELER-JOHNSON TEST (TEST FOR PYRIMIDINES)REAGENTS: Reagents: sat’d. Br2-H2O, Ba(OH)2
(+) VISIBLE RESULT: purple coloration
PRINCIPLE INVOLVED:
1) formation of dialuric acid
2) neutralization
TEST FOR: PYRIMIDINES (CYTOSINE, URACIL) (T is negative)
D. MUREXIDE TEST (TEST FOR PURINES)REAGENTS: concentrated HNO3, NH4OH/KOH
(+) VISIBLE RESULT: Purple-red color solution
PRINCIPLE INVOLVED:
1) oxidation of purine forming dialuric acid & alloxan
2) condensation reaction forming alloxanthin
3) neutralization reaction producing purple-red murexide or ammonium
purpurate
TEST FOR: PURINES (ADENINE, GUANINE)
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E. PHOSPHATE TESTREAGENTS: Concentrated HNO3, (NH4)2MoO4
(+) VISIBLE RESULT: Yellow precipitate or crystals
PRINCIPLE INVOLVED:
(NH4)3PO4 12 MoO4
TEST FOR: PHOSPHATE