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Nucleic Acid Experiment

This document describes experiments involving the isolation and analysis of nucleic acids. It discusses isolating RNA from yeast by heating with NaOH to lyse cells and extract nucleic acids. It also describes tests to detect the presence of phosphates, pentoses, purines, and nucleoproteins in nucleic acid samples. The document explains the roles of various reagents like sulfuric acid and ethanol in isolating RNA and hydrolyzing nucleic acids into nucleotides. It also outlines the biological functions of different types of nucleic acids like DNA, mRNA, tRNA, and rRNA.

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37 views2 pages

Nucleic Acid Experiment

This document describes experiments involving the isolation and analysis of nucleic acids. It discusses isolating RNA from yeast by heating with NaOH to lyse cells and extract nucleic acids. It also describes tests to detect the presence of phosphates, pentoses, purines, and nucleoproteins in nucleic acid samples. The document explains the roles of various reagents like sulfuric acid and ethanol in isolating RNA and hydrolyzing nucleic acids into nucleotides. It also outlines the biological functions of different types of nucleic acids like DNA, mRNA, tRNA, and rRNA.

Uploaded by

jvlegaspi7463val
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BIOCHEM MIDTERM

CHEM113
1st
EXPERIMENT

NUCLEIC ACID EXPERIMENT Bial’s reagent


1. Orcinol: Orcinol is an organic compound and is the
Nucleic acids are complex organic compounds composed of primary reagent responsible for the reaction in the
nucleotides. Each nucleotide is composed of a pentose Bial's test. It reacts with pentoses and specific
sugar, a nitrogen base, and phosphate. Depending on the hexoses to produce colored products.
pentose sugar present, two kinds of nucleic acids are 2. Concentrated Sulfuric Acid: Concentrated sulfuric acid
known: DNA whose sugar is deoxyribose, and RNA with is used to create an acidic environment, which is
ribose sugar. Qualitative analyses of nucleic acids based necessary for the reaction with orcinol to occur. The
on the presence of phosphorus as phosphate, pentose acid catalyzes the reaction and helps in the formation
sugar, and the nitrogen which are derivatives of purine and of the characteristic color.
pyrimidine. 3. Water: Water is added to dilute the reagent and make
it ready for use in the test
Isolation of RNA from Yeast
The process of the isolation of RNA from yeast, Test for Purines
Saccharomyces cerevisiae, involves heating with NaOH
which served to disrupt the cell membrane and lyse the cell Add excess 2M ammonia solution and a few drops of 0.1M
to be able to extract the nucleic acids from the said test silver nitrate to 1 mL of DNA extract. A white precipitate
sample. The NaOH increases the pH level of the solution indicates the presence of purines. The precipitation
resulting in the denaturation of contaminant proteins, mechanism is by a reaction of Ag+ with the nitrogens of
inactivates nucleases which can degrade RNA. Heating purines.
helped loosen the cell membrane by increasing the kinetic NH4OH 1%, AgNO3
energy of the lipid molecules, making it release more RNA.
QUESTIONS

Test for Nucleoprotein 1. Role of Reagents in the Isolation of RNA:

The mixture of isolated RNA sample with NaOH and 1% of A. 1% NaOH (Sodium Hydroxide): Sodium hydroxide is
copper have resulted a different color as it was when mixed used to lyse cells and tissues in RNA isolation. It disrupts
properly. While shaking the mixtures, research have the cell membranes and denatures proteins, including
shownthat NaOH destroys the RNA completely in 10-15 ribonucleases, which helps protect RNA from degradation
minutes of mixing. On the other hand, the copper added to during the isolation process.
NaOH you will get a precipitate of copper (II) hydroxide,
Cu(OH). Which can be seen formed in a mixture of the 3 B. Acetic Acid: Acetic acid is often used to neutralize the
substances. effects of sodium hydroxide. After cell lysis with NaOH,
• A positive result in test for nucleoproteins will acetic acid is added to lower the pH and stabilize the RNA.
yield a gray precipitate. This step is crucial in RNA isolation.

Mild Acid Hydrolysis C. 95% Ethanol: Ethanol is used to precipitate RNA from
the aqueous phase of a sample. RNA is not soluble in high
The substance will appear quite clearer as it did after the concentrations of ethanol, and it can be recovered by
boiling of RNA and sulfuric acid as it became more centrifugation or other separation techniques.
concentrated.
2. Role of 10% H2SO4 in RNA Hydrolysis:
10% H2SO4
Test for Phosphate 10% H2SO4 (sulfuric acid) is used in the hydrolysis of
RNA to break it down into its constituent nucleotides.
Phosphate reacts with concentrated nitric acid and Sulfuric acid is a strong acid and, when used in the
ammonium molybdate to form yellow precipitate of hydrolysis process, it cleaves the phosphodiester bonds
ammonium phosphate molybdate. between the ribonucleotides, releasing them as individual
nucleotides.
Confirmatory test of phosphate using magnesia mixture
text: Phosphate reacts with magnesia mixture to form white 3. Biological Functions:
precipitate of magnesium ammonium.
Ammonia water, 6N HNO3, (NH4)2 MoO4 solution a. DNA (Deoxyribonucleic Acid): DNA stores genetic
information in all living cells. It serves as a blueprint for the
Test for Pentose synthesis of RNA and proteins, playing a fundamental role
in the transmission of genetic traits from one generation to
Reacts with the orcinol to generate a colored substance. The
the next.
solution will turn bluish and a precipitate may form to
determine the presence of Pentose (ribose). b. mRNA (Messenger RNA): mRNA carries genetic
information from DNA to the ribosome, where it serves as a
The color change from greenish-blue to red indicates a
template for protein synthesis. It conveys the genetic code,
positive test for the presence of ribose. This change in
specifying the amino acid sequence in a protein.
color is due to the reaction of ribose with Bial's reagent to
form a complex that absorbs light in the visible spectrum.
BIOCHEM MIDTERM
CHEM113
1st
EXPERIMENT

c. tRNA (Transfer RNA): tRNA is involved in protein


synthesis by bringing amino acids to the ribosome. Each
tRNA molecule is specific for a particular amino acid, and it
interprets the codons on mRNA to assemble the correct
amino acids in a growing polypeptide chain.

d. rRNA (Ribosomal RNA): rRNA is a structural component


of ribosomes, which are the cellular organelles responsible
for protein synthesis. It catalyzes the formation of peptide
bonds between amino acids during translation.

4. Products of Nucleic Acid Hydrolysis:

The hydrolysis of nucleic acids, whether RNA or DNA,


yields the following products:

- Nucleotides: The individual building blocks of nucleic


acids, consisting of a nitrogenous base, a sugar (ribose in
RNA and deoxyribose in DNA), and a phosphate group.

- Nitrogenous Bases: Purines (adenine and guanine) and


pyrimidines (cytosine, thymine in DNA, and uracil in RNA)
are released during hydrolysis.

5. DNA Hydrolysis by Alkali:

DNA can be hydrolyzed by alkali, such as sodium


hydroxide (NaOH), in a process called depurination. In this
process, the alkali breaks the glycosidic bond between the
deoxyribose sugar and the nitrogenous base (purine or
pyrimidine). However, it's important to note that this
reaction specifically targets the glycosidic bond and not the
phosphodiester backbone of DNA. The cleavage of the
glycosidic bond can lead to the loss of the nitrogenous
base. This is a common method used to quantify DNA
content in a sample or to remove RNA contamination.

COMPOUNDS

• H2SO4: Sulfuric Acid


• HNO3: Nitric Acid
• (NH4)2MoO4: Ammonium Molybdate
• NH4OH: Ammonium Hydroxide (or Ammonia
Solution)
• AgNO3: Silver Nitrate

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