E2 - SECONDARY STRUCTURE OF DNA

Genetic material found in the nucleus

DNA

Makes copy of itself during cell division

DNA

Provide template for RNA biosynthesis or transcription

DNA

Transcribes the genetic information in DNA during RNA biosynthesis or transcription

RNA

Carries and expresses genetic information transcribed via protein biosynthesis or translation in the ribosomes

RNA

DNA structure

Double stranded and Anti-parallel

Components of DNA

Nucleotide, Sugar, Phosphate

Absorbance measurement are measured in these wavelengths

260, 280, and 230

Nucleic acids are seen at this wavelength

260 nm

What is seen in wavelength 260 nm

Presence of aromatic nitrogenous bases (purines and pyrimidines)

Effect of denaturation in absorbance reading in 260 nm

Higher reading due to hyperchromic effect (helix unwinding)

Primary contaminants of Nucleic acids

Proteins

Proteins are seen at what wavelength

280 nm

What is seen in 280nm

Aromatic amino acids Trp, Phe, and Tyr

How to compute for the concentration of DNA

Relative purity can be determined using

A260/A280

Range for good quality nucleic acid in A260/280

1.8 - 2.0

Pure DNA at A260/280

1.8

A260/280 reading lower than 1.8 indicates

Increased contamination of protein

A260/280 reading higher than 1.8 indicates

Contamination by RNA or denatured DNA

Assess the effect of changes in pH on the structural integrity of DNA, reflecting helix-to-coil transition

Viscometry

Factors affecting viscosity in this experiment

Moelcular size/shape and DNA concentration

Formula for Viscosity

Formula for relative viscosity

Formula for intrinsic viscosity

Phosphate group binds to one nucleotide to another through

condesation reaction

bond of phosphate and nculeotide

phosphodiester bond 3’ 5’

why is it challenging to extract DNA compared to proteins

DNA is usually packed in a compact space in the nucleus, deep inside the cell

Why is it difficult to extract DNA completely intact

It is a very long and fragile biomolecule

Factors affecting DNA stabilizing

1. pH

2. Temperature

3. Ionic stregth

4. Cellular conditions

   1. Mechanical stress

What does pH stabilize

Nucleotides

Ideal pH of H bonds

pH 4 - 10

Ideal pH of Phosphodiester linkages

pH 3 - 12

Ideal pH of N-glycosidic bonds to purine bonds

> pH 3

Temperature where DNA unwinds

80 - 90 C

Temperature where phosphodiester linkages and N-glycosidic bonds breask

Higher than 100C

Ideal ionic stregths for DNA

< 0.05 M weakens H-bonding between complementary strands of DNA

Factors to look into lysis protocol

DNA nucleases must be deactivated and other contaminants must be seperated

Detergents use

Lysing certain membranes and denatrues enzymse

Proteinases use

Degrage nucleases

Chelating agents use

Sequester the metal cofactor of nucleases to deactivate them

RNAses use

Degrade RNA to lower RNA contamination

Where should DNA be stored

In a solution

What happens to a dry DNA

undergoes denaturation

Important thing to consider for mechanical stress

Grinding, shaking, stirring may cause cleavage (shearing or scisson) of DNA chains making the isolated strands shorter or damaged

General protocol for DNA isolation

• Lysis/Homogenization

• Removal of contaminatns

• Precipitation of DNA

• Storage

How is DNA precipitated

Adding ice-cold ethanol or isopropyl alcohol

Solution used to store DNA

SSC buffer

Why is SSC buffer used

• Maintains DNA stability

• Reduces ionic interaction of histones and DNA backbone

  • Has lower pH to maintain positive charge of histones

Temperature ideal for protein denaturaing

60 C

SSC buffer pH

8

Why is SDS used

protein denaturation, disruption of ionic interaction betwen histones and DNA, inactivated of enzymes

NaCl use

Helps competely dissociate DNA-histone complex and percipitation by increasing inonic strength

Meat tenderizer use

Proteinase, deactivating nucleases

How does nuclease degrade DNA

Cleaves phosphodiester bond

Use of isoamyl alcohol or phenol-chlorofom

Improve phase separation between organic and aqueous layers and remove polar contaminants

EDTA use

Inactivates remaining enzymes, chelates Mg and Ca

Cold alcohol use

Precipitation of DNA, lowers dielectric constant of the solvent by introducing a lower temperature, lowering solubility of DNA

Importance of cold temperatures

Preserves DNA and minimize enzymatic activity

How to compute for protein concentration

Principle for 320 measurement

Turbidity correction

Purity measurement with turbidity correction

What structure of DNA correlates to decreased viscosity

Coiled DNA

What happens when DNA is neutralized

Denatured DNA should refold back into a double helix

What happens to the viscosity of DNA after adding denaturants

Decreaes