Nucleic Acids and Nitrogenous Bases

Questions and Answers

What is the primary purpose of heating with alkali during yeast RNA isolation?

To denature proteins and prevent RNA degradation

What is the function of acid extraction at pH 4-5 in yeast RNA isolation?

To separate nucleic acids from associated proteins

What is the purpose of treating with alcohol and conc. HCl during yeast RNA isolation?

To precipitate RNA

What is the purpose of repeated washings with alcohol and ether/organic solvents during yeast RNA isolation?

To remove lipids and other interfering substances Signup and view all the answers

What is the primary reason for avoiding drastic changes in nucleic acid structures during isolation?

To maintain the integrity of the nucleic acid structure Signup and view all the answers

What is the general procedure for isolating nucleic acids?

Disruption of the cell membrane, treatment with a solution to dissociate nucleoprotein, and purification of nucleic acids Signup and view all the answers

What is the primary basis for obtaining intact DNA from a solution of DNA and RNA?

Differential solubility of DNA and RNA in ethanol Signup and view all the answers

What is the function of adding ethanol to a solution of DNA and RNA?

To precipitate out the DNA Signup and view all the answers

What is the purpose of the acid hydrolysis step in the isolation of RNA?

To break down the RNA into its constituent nucleotides Signup and view all the answers

What is the result of adding AgNO3 to the hydrolyzed RNA sample?

A whitish precipitate Signup and view all the answers

What is the purpose of the centrifugation step in the isolation of RNA?

To separate the RNA from the supernatant Signup and view all the answers

What is the result of Benedict's test on unhydrolyzed RNA?

A positive result due to the presence of ribose sugars Signup and view all the answers

What is the purpose of the cheese cloth filtration step in the isolation of RNA?

To filter out impurities from the RNA solution Signup and view all the answers

What is the primary difference between the qualitative tests for the hydrolyzed and unhydrolyzed RNA samples?

The difference in color of the precipitate Signup and view all the answers

What is the purpose of adding glacial acetic acid to the supernatant liquid?

To lower the pH of the solution until it becomes faintly acidic Signup and view all the answers

What is the result of heat treatment for 15 minutes with occasional stirring?

Breakdown of yeast cells Signup and view all the answers

What is the purpose of centrifugation and decantation in the RNA isolation process?

To separate the RNA from the impurities Signup and view all the answers

What is the composition of the initial solution used for RNA isolation?

5mL 1% NaOH + 25mL H2O + 5.0g dry yeast Signup and view all the answers

What is the purpose of cooling the solution to 40°C or lower?

To prevent degradation of RNA Signup and view all the answers

What is the purpose of adding 0.2mL of conc. H2SO4?

To precipitate the RNA molecules Signup and view all the answers

What is the purpose of evaporating the solution over a water bath?

To reduce the volume of the solution to 10mL Signup and view all the answers

What is the final step in the RNA isolation process?

Weighing the dry crude extract and calculating yield in g/mL Signup and view all the answers

Study Notes

Nitrogenous Bases and Nucleic Acids

Nucleic acids are major components of all cells, making up 5-15% of their dry weight.
Three types of interactions maintain the rigid molecular configuration of nucleic acids: phosphodiester bonds, hydrogen bonds, and van der Waal's forces.
Isolation of nucleic acids should be conducted to avoid drastic changes in their structures.

Characteristics of Yeast (Saccharomyces cerevisiae)

Yeast is a unicellular fungus.
It contains 4% RNA by weight.

Isolation of Yeast RNA

The isolation process involves heating with alkali (NaOH) to extract nucleic acids and water-soluble proteins, and inactivating nucleases that degrade RNA.
The nucleic acid is then separated from associated proteins and other interfering substances by acid extraction at pH 4-5.

Steps in Isolation of Yeast RNA

Mix 5mL 1% NaOH with 25mL H2O and 5.0g dry yeast, then heat for 15 minutes with occasional stirring.
Strain the mixture through cheesecloth/gauze and collect the supernatant liquid.
Add glacial acetic acid until the solution is faintly acidic.
Centrifuge and decant the solution, then evaporate it over a water bath until the volume is 10mL.
Repeat the process until a clear liquid is obtained.
Cool the solution to 40°C or lower and set it aside.

Qualitative Analysis of RNA from Yeast

Test for guanine or adenine by adding conc. HCl and NH4 to the hydrolyzed sample, resulting in a whitish precipitate.
Test for inorganic phosphate by adding (NH4)2MoO4 to the hydrolyzed sample, resulting in a yellow precipitate.
Conduct Benedict's test to detect reducing sugars in unhydrolyzed RNA.

Objectives and Apparatus

Objectives: isolate RNA from yeast, conduct acid hydrolysis, and conduct qualitative tests on both hydrolyzed and unhydrolyzed RNA.
Apparatus: beakers, stirring rod, dropper/pipette, cheesecloth, graduated cylinder, test tubes, litmus paper, hot plate, and centrifuge.

Analysis and Guide Questions

Guide questions:
- How can intact DNA be obtained from a solution of DNA and RNA?
- How can you distinguish purines from pyrimidines via hydrolysis procedures?
- Compare the results of the qualitative tests for the hydrolyzed and unhydrolyzed samples.

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Description

Learn about the different components of nucleic acids, including nitrogenous bases, and the interactions that give them their structure. This quiz covers the basics of nucleic acids and their composition.