Nucleic Acids

Experiment 4

Experiment No. 4 NUCLEIC ACIDS

Function:

storage and transmission

of genes
2 types: DNA & RNA
Components:
A] Nitrogenous Bases (nucleobase)
B] Sugars (ribose or deoxyribose)
C] Phosphates

Nucleoprotei
ns
Nucleic Acids
made up of
mononucleotid
e
Phosphate
(PO4-3)

Histones
Proteins rich in
basic aa

Nucleosides

Base

Sugar

Bases

Pyrimidine

Purine

Sugars of Nucleic Acid

Nucleosides = Base + Sugar

Nucleosides
Ribonucleosides

Deoxyribonucleosides

Cytidine

Deoxycytidine

--

Deoxythymidine

Uridine

--

Adenosine

Deoxyadenosine

Guanosine

Deoxyguanosine

Nucleotides
Ribonucleotides

Deoxyribonucleotides

Cytidine
5monophosphate

Deoxycytidine
5monophosphate

--

Deoxythymidine
5monophosphate

Uridine
5monophosphate

--

Adenosine
5monophosphate

Deoxyadenosine
5monophosphate

Guanosine
5monophosphate

Deoxyguanosine
5monophosphate

Base + Sugar + Phosphate =

NUCLEOTIDE

Properties of
Nucleic Acids
Absorbs light
Exists in

tautomeric forms

Nucleic Acids are nucleotide polymers

Two types:
1. Deoxyribonucleic
Acid (DNA)
2. Ribonucleic Acid
(RNA)

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.

ISOLATION & IDENTIFICATION OF DNA

Qualifications of a good DNA source:
1] Tissue has low DNase activity
2] Low ratio of other cell constituents to
DNA
3] Weak attraction between the DNA & the
protein
4] DNA is readily obtainable & available in
essentially pure form
Tissues that are best source of DNA:
Thymus, spleen, liver, pancreas

General steps involve in

isolation & purification of DNA
1] Disruption of cells & membrane-bound
structures to release DNA
2] Inactivation of enzymes that hydrolyze
the
DNA
3] Dissociation & denaturation of proteins
4] Solvent extraction & concentration of
the
DNA by precipitation

HOMOGENIZATION
Disruption

of cell membranes & release of DNA into

medium
Homogenizing agent : 0.1 M NaCl - 0.5 M sodium citrate,
pH 7.5
NaCl provides isotonic environment dissolves

deoxyribonucleoprotein stabilizes the DNA

Sodium citrate inactivates DNase (by removing Ca2+ & Mg2+
cofactors)
pH 7.5 inactivates DNase. denature some proteins, loosens bond
between histones & DNA
SDS emulsifies cell lipids/proteins causing cell membrane to
breakdown; disrupts polar interaction that hold the cell membrane
together
CHCl3-isoamyl

alcohol - removes protein from the solution

CHCl3 causes surface denaturation of proteins

Isoamyl alcohol stabilizes interface between organic & aqueous

phases to reduce foaming

 CENTRIFUGATION
For separating & analyzing cells, organelles, & biological
macromolecules

centrifuge (0 C - 10C) prevents

denaturation of DNA

Refrigerated

95% Ethanol precipitate out DNA

Ribonuclease

purifies DNA isolate

ULTRAVIOLET MEASUREMENT of isolated nucleic

acids
1.6 1.8 pure DNA
1.9 2.0 pure RNA
low ratio a lot of protein contamination

ISOLATION OF RNA
Qualifications of a good RNA source:
1] high cytoplasmic/nuclear ratio
2] low RNase activity
3] RNA should be easily purified
4] readily available

Purification begins with solvent extraction that

denatures proteins & inactivates cellular
ribonuclease
1% NaOH extracting solvent, inhibits RNase,

denatures proteins
Glacial

HOAc ppt. out proteins

95% EtOH ppt. RNA

DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not?
Vicinal -OH groups (2' and 3') in
RNA make it more susceptible to
hydrolysis
DNA, lacking 2'-OH is more stable
This makes sense - the genetic
material must be more stable
RNA is designed to be used and
then broken down

Hydrolysis of Nucleic
Acids
RNA

is resistant to dilute acid

DNA is depurinated by dilute acid
DNA is not susceptible to base
RNA is hydrolyzed by dilute base

HYDROLYSIS OF NUCLEIC
ACIDS
N-glycosidic

purine & pyrimidine linkages = stable to mild acidic

& basic conditions
With basic hydrolysis (37C w/ 0.3 M KOH)
phosphodiester linkages of RNA are cleaved forming
2 & 3-phosphoribonucleotides or 2,3-cyclic
monophosphonucleotides
1 structure of DNA stable

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
With

stronger acids at higher T (1N TCA, HCl, HClO4; 100C, 15

mins.)
purine bases of DNA are cleaved from
2-deoxyribose = depurination

Restriction Enzymes
Bacteria

have learned to "restrict"

the possibility of attack from foreign
DNA by means of "restriction
enzymes"
Type II and III restriction enzymes
cleave DNA chains at selected sites
Enzymes may recognize 4, 6 or more
bases in selecting sites for cleavage
An enzyme that recognizes a 6-base
sequence is a "six-cutter

CHEMICAL
CHARACTERIZATION
A] Test for deoxybibose
(DischeRxn./Diphenylamine Test)
Reagents: Diphenylamine, glacial HOAc,
conc. H2SO4
(+) result: Blue complex
Principle: 1] dehydration of deoxyribose forming
-hydroxylevulinaldehyde, 2] complexation
reaction w/ diphenylamine
B] Test for phosphate
Reagents: conc. HNO3, (NH4)2MoO4
(+) result: yellow crystals
Principle: (NH4)3PO4 12 MoO4

CHEMICAL
CHARACTERIZATION
C] Test for Purines (Murexide Test)
Reagents: conc. HNO 3, NH4OH/KOH
(+) result: purplish red solution
Principle: 1] oxidation of purine forming dialuric
acid & alloxan
2] condensation reaction forming
alloxanthin
3] neutralization reaction producing
the purple-red murexide or
ammonium purpurate

CHEMICAL
CHARACTERIZATION
D] Test for pyrimidines (Wheeler-Johnson Test
For C or U (T is negative)
Reagents: satd. Br2-H2O, Ba(OH)2
(+) result: purple coloration
Principle: 1] formation of dialuric acid
2] neutralization
E] test for ribose (Bials-Orcinol Test)
Specific for pentoses
Reagents: Orcinol, FeCl3, HCl
(+) result: bluish-green solution
Principle: dehydration forming furfural &
condensation with orcinol

DETERMINATION OF BASE
COMPOSITION BY PAPER
CHROMATOGRAPHY
Solvent

system: Isopropanol-conc. HCl-

H 2O
(65:16:718.5)
0.1N HCl:9.17 mL H 2O:0.83 mL conc. HCl
% mg each of A, G, C, & T in 2 mL 0.1N HCl
Approximate Rf values:

0.36
G 0.25
C 0.47
T 0.77