EXPERIMENT 4

NUCLEIC ACIDS
I. NUCLEIC ACIDS
Function: storage and transmission of genes
2 types: DNA & RNA
3 Components:
Nitrogenous Bases (nucleobase)
Sugars (ribose or deoxyribose)
Phosphates
Solubility of Nucleic Acids
1. DNA is soluble in H2O
2. 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/RNA
High pH (11-12) = ssDNA is stable; RNA is degraded
Low pH = DNA bases are removed (apyrimidic or apurinic sites) bec glycosidic bonds
are cleaved.
II. ISOLATION
General 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 DNA
3. Dissociation & denaturation of proteins
4. Solvent extraction & concentration of the DNA by precipitation

Reagents used

Purpose

1. Homogenizing solution:
a. SDS
(sodium
sulphate)

dodecyl

emulsifies cell lipids/proteins causing cell membrane

to breakdown; disrupts polar interaction that hold the
cell membrane together

b. EDTA
(ethylenediamminetetraacetic
acid)
c. NaCl

Chelates Ca2+ and Mg2+ cofactors leading to

inactivation of DNase

d. Sodium citrate

buffer

2. Meat tenderizer

provides
isotonic
environment
dissolves
deoxyribonucleoprotein stabilizes the DNA

Hydrolyses proteins that can contaminate DNA

(contains bromelain)
3. Ice cold 95% ethanol

III.

Prepitates nucleic acids

The cold environment decreases the solubility of
nucleic acids facilitating faster precipitation.

HYDOLYSIS OF NUCLEIC ACIDS

A. 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
nucleoside
1. 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 TESTS
A. BIALS 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 -hydroxylevulinaldehyde

2) complexation reaction w/ diphenylamine

TEST FOR:

DEOXYRIBOSE

C. WHEELER-JOHNSON TEST (TEST FOR PYRIMIDINES)

REAGENTS:
Reagents: satd. Br2-H2O, Ba(OH)2
(+) VISIBLE RESULT:

purple coloration

PRINCIPLE
INVOLVED:

1) formation of dialuric acid

TEST FOR:

PYRIMIDINES (CYTOSINE, URACIL)

2) neutralization
(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 TEST
REAGENTS:

Concentrated HNO3, (NH4)2MoO4

(+) VISIBLE RESULT:

Yellow precipitate or crystals

PRINCIPLE
INVOLVED:

(NH4)3PO4 12 MoO4

TEST FOR:

PHOSPHATE