experiment 4

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

(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

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)

E. PHOSPHATE TESTREAGENTS: Concentrated HNO3, (NH4)2MoO4

(+) VISIBLE RESULT: Yellow precipitate or crystals

PRINCIPLE INVOLVED:

(NH4)3PO4 12 MoO4

TEST FOR: PHOSPHATE