PHA6112 LAB: NUCLEIC ACIDS PRE & POST LAB

Nucleic acids

Biomolecules that store information for cellular growth and reproduction

False: DNA stores; RNA synthesizes

T or F: RNA stores the information while the DNA synthesizes the proteins

proteins, carbohydrates, lipids, and nucleic acids

The proteins produced in the cell are needed to make other ______, _______, _______, and ______.

Nitrogen heterocyclic base and Pentose sugar

What are the 2 parts of a nucleoside?

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA)

2 Types of Nucleic acid

nucleotides

These are polymers consisting of long chains of monomers called _______.

Nitrogen heterocyclic base, Pentose sugar, and Phosphate residue

What are the 3 parts of nucleotide?

Isolation of Microbial DNA

What isolation is this?

Isolation of Plant DNA

What isolation is this?

Isolation of DNA from Onion

What isolation is this?

Isolation of DNA from Animal Tissue

What isolation is this?

Isolation of RNA from Yeast

What isolation is this?

Acid Hydrolysis of DNA

Test for Deoxyribose Test for Phosphate Test for Purines

Test for Pyrimidines

Alkaline Hydrolysis of RNA

Test for Ribose

Test for Phosphate

Test for purines

Test for Pyrimidine

Test for Ribose: Bial's Orcinol Test

Reagent: Orcinol in HCL (yellow solution

Positive result: blue-green coloration

Test for Deoxyribose: Diphenylamine or Dische (1930) test

Reagent: diphenylamine in conc. H2 SO4

Positive result: : blue complex/compound (λmax = 595 nm)

Test for Phosphates

Reagent: Sulfuric acid, conc. HN03, and 10% (nh4)mOo4

Positive result: yellow crystalline precipitate

Test for Purines: Murexide Test

Reagent: NA solution, conc. HN03, 10% KOH

Positive result: red residue

Adenine and guanine

What are the two derivatives of the test for purine?

Test for Pyrimidines

Reagent: NA solution, bromine water, Ba(OH)2 solution

Positive result: purple color due to Ba2+ salt of dialuric acid

True

T or F: Test for pyrimidines detects U and C

False: Negative for T

T or F: Test for pyrimidine is negative for G

True

T or F: Each nucleotide unit is made up of nitrogen heterocyclic base, pentose sugar, and phosphate residue.

Deoxyribose

The pentose sugar in DNA contains what?

Ribose

The pentose sugar in RNA contains what?

Pyrimidine

6-membered heterocyclic bases

Purine

Fused 6- & 5-membered rings for its heterocyclic bases

Pyrimidine

Heterocyclic base that contains cytosine, uracil, and thymine

Purine

Heterocyclic base that contains adenine and guanine

RNA

Uracil is found in ____.

DNA

Thymine is found in ___.

Cytosine

A pyrimidine base that contains an amine group

Thymine

A pyrimidine base that contains a methyl group

Guanine

A purine base that contains a carbonyl group

tissues containing cells with high nuclear volume/cytoplasmic volume ratio

choice of sample in isolation of DNA

Bacillus subtilis or Escherichia coli

Microbial DNA sample source in isolation of DNA

meristematic region of any plant & yellow onion

Plant DNA sample source in isolation of DNA

spleen, liver, thymus and pancreas (vs brain and muscle tissues - lower concentration)

Animal DNA sample source in isolation of DNA

1. Homogenization

2. Dissociation and denaturation/precipitation of nucleoprotein

3. Purification of NA

general principle and steps in the isolation of DNA

homogenization

lysis or disruption of cell membrane and organelle membranes releases the nucleoprotein (DNA-histone complex) into a medium in which it is soluble and protected from degradation

Mechanical disruption

homogenization using homogenizer, blender, mortar and pestle, cut into small/fine pieces or mashed, mincing, grinding, sonication

Chemical

homogenization using detergents & chaotropic agents

n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea

examples of detergents & chaotropic agents

to inactivate nucleases

why is denaturation of enzymes performed?

salt, alcohol (isopropyl alcohol or ethanol)

reagents used in dissociation and denaturation/precipitation of nucleoprotein

T

T or F: it is difficult to isolate DNA in an intact and undamaged form because of its large and fragile nature

F (drastic changes in conditions are avoided or minimized)

T or F: Isolation should be conducted where drastic changes in conditions (experimental factors affecting DNS) are observed

isolation of microbial DNA

Target for extraction is the disruption of bacterial cell wall and the inactivation of enzymes

EDTA (Ethylenediaminetetraacetic acid)

in the isolation of microbial DNA, it is used as chelating agent for divalent cations (Mg++) for the metals present in DNases. This inactivates the enzyme.

Lysozyme

in the isolation of microbial DNA, it is used to cause lysis of bacterial cells by hydrolyzing the peptidoglycan present in cell walls. This disrupts the bacterial cell wall to let the DNA be in solution

plant DNA

One of the most difficult DNAs to isolate

• Structure of the plant cell - hard, rough, solid cell wall because of peptidoglycan, pectin, cellulose and chitin.

• Component of the plant cell - secondary metabolites such as polysaccharides, phenols etc.

reasons why plant DNA is difficult to isolate

CTAB (Cetyl trimethylammonium bromide)

in the isolation of plant DNA, it is a cationic detergent used to separate polysaccharides during purification of DNA samples from plants

NaCl

in the isolation of plant DNA, it removes proteins that are conjugated to the DNA. Proteins are kept dissolved in the aqueous portion which prevents alcohol from precipitating it along with DNA.

0.15 M NaCl, 5% SDS (sodium dodecyl sulfate), 0.15 M sodium citrate, 1 mM EDTA

components of homogenizing solution

0.15 M NaCl

component of homogenizing solution that precipitates nucleoproteins (salting-out)

5% SDS (sodium dodecyl sulfate)

component of homogenizing solution that breaks ionic interaction between protein and nucleic acid

0.15 M sodium citrate and 1 mM EDTA

component of homogenizing solution that Chelate Ca2+ and Mg2+ ions (cofactors)

dissolves nucleic acids

Heating at 60°C

retards nuclease activity

Cooling in ice bath

denatures protein

Papain or meat tenderizer 6%

precipitates DNA and RNA

Ice-cold 95% EtOH

Precipitates DNA only

Ice-cold 100% iPrOH

5 M Sodium chloride

Lysing buffer: osmosis in the cell

Tris HCl

Lysing buffer: buffer

0.5M EDTA

Lysing buffer: chelates the metals in the DNAses

5% SDS

Lysing buffer: disrupts cell membrane and nuclear envelope

Chloroform

denature the proteins and lipids to maintain separation of organic and aqueous phases

Isoamyl alcohol

prevents foaming

F (Large cytoplasmic volume)

T or F: RNA is single-stranded NA found in high concentrations in tissues with low cytoplasmic volume

T

T or F: RNA isolates are easily damaged by shearing

T

T or F: RNA is very vulnerable to digestion by ribonucleases (endogenous and exogenous)

F (higher UV absorption)

T or F: RNA has lower UV absorption at 260 nm than DNA

T

T or F: Isolation of RNA from Yeast (S. cerevisiae) has low nuclear-cytoplasmic volume ratio

T

T or F: RNA concentration is high in the cytoplasm

F (4% RNA by weight)

T or F: Saccharomyces cerevisiae is 10% RNA by weight

Heating with dilute NaOH;

Glacial acetic acid (pH 4-5);

Ethanol with concentrated HCl;

Alcohol (EtOH) and ether (organic solvents);

TE Buffer;

SSC (Standard Saline Citrate) Solution

processes and reagents involved in Isolation of RNA from Yeast (S. cerevisiae)

Heating with dilute NaOH

Isolation of RNA from Yeast (S. cerevisiae)

—Separates RNA from proteins

—Extracts RNA and water-soluble proteins

—Inactivates nucleases (RNase)

Glacial acetic acid (pH 4-5)

Isolation of RNA from Yeast (S. cerevisiae)

Separates nucleic acids associated with proteins and other interfering substances

Ethanol with concentrated HCl-

Isolation of RNA from Yeast (S. cerevisiae)

precipitates RNA

Alcohol (EtOH) and ether (organic solvents)

Isolation of RNA from Yeast (S. cerevisiae)

Removes lipids

TE Buffer, SSC (Standard Saline Citrate) Solution

Isolation of RNA from Yeast (S. cerevisiae)

preserves the integrity of DNA by maintaining the pH of the solution

260, 280, and 230 nm

What are wavelengths in measuring absorbance of nucleic acids? (3)

A260/280

relative measure of NA/protein content

1.8-2.0

range of relative measure for good quality NA

1.8

relative measure of pure DNA

indicates increased contamination by protein

what does a relative measure of x < 1.8 indicate?

indicates increased contamination by RNA or denature DNA

what does a relative measure of 1.8 < y indicate?

A = ԑbc

formula for Beer-Lambert's Law

Produces brown-black solid

visual positive result of Acid hydrolysis

F (not complete for RNA)

T or F: Alkaline (basic) Hydrolysis is not complete for DNA

Alkaline (basic) Hydrolysis

what type of hydrolysis creates a mixture of 2' and 3'- nucleotides

F (DNA is not readily hydrolyzed by dilute alkali)

T or F: RNA is not readily hydrolyzed by dilute alkali