BIOCHEMISTRY LAB MANUAL (NUCLEIC ACIDS)

GROUP 2 - 1NU23
Members:
CACHO, Ellabel Genestralin Gabriel B.
CARPIO, Laura Ashley A.
CLAVER, Alexandre James S.
CRUZ, Danelle S.
DE OCAMPO, Nicole Anne M.

PAGE 73
ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS (DNA)

1. State three major differences between the structures of DNA and RNA.

Firstly, the sugar component in their structures is distinct. DNA contains deoxyribose sugar,
while RNA contains ribose sugar. Secondly, their nitrogenous bases vary. Both DNA and RNA
have four bases each, but DNA includes adenine (A), cytosine (C), guanine (G), and thymine
(T), whereas RNA replaces thymine with uracil (U). Lastly, their structures differ in terms of
shape. DNA typically forms a double-stranded helix, while RNA is usually single-stranded.

2. Give at least two differences between prokaryotic and eukaryotic DNAs.

Prokaryotic DNA is a single, circular chromosome located in the nucleoid region of the cell,
without the protection of a nucleus. In contrast, eukaryotic DNA is organized into multiple linear
chromosomes inside a nucleus.
Eukaryotic cells have membrane-bound organelles like the nucleus and mitochondria, each
with its own DNA. Prokaryotic cells lack these organelles and don't have specialized DNA for
them.

3. What is the range of expected A260/A280 ratio for pure DNA isolates?

The expected A260/A280 ratio for pure DNA isolates typically falls in the range of 1.8 to
2.0. This ratio is commonly used to assess the purity of DNA samples. A ratio within this range
suggests that the DNA is relatively free from contamination by proteins, RNA, or other impurities
that can absorb light at different wavelengths.

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ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS (DNA)

1. Explain why the following reagents are used in the isolation of DNA:
A. MICROBIAL DNA ISOLATION
 I. EDTA
EDTA, or ethylenediaminetetraacetic acid, is employed in microbial DNA isolation due to
its ability to chelate divalent cations, primarily magnesium (Mg2+), which are cofactors for
enzymes like nucleases. By sequestering these cations, EDTA inhibits the activity of nucleases,
preventing them from degrading the DNA during the isolation process. This crucial step ensures
the preservation of DNA integrity, which is essential for obtaining high-quality microbial DNA
samples suitable for various downstream applications, including PCR, sequencing, and genetic
analysis, ultimately enabling accurate and reliable results.

II. Lysozyme
Lysozyme is utilized in microbial DNA isolation because it is an enzyme capable of
breaking down the bacterial cell wall by hydrolyzing the glycosidic bonds in peptidoglycan, a
major component of the cell wall in many bacteria. By effectively weakening and disrupting the
cell wall, lysozyme helps to release the cellular contents, including the microbial DNA, into the
surrounding solution. This initial step in the DNA isolation process is essential for liberating the
DNA from the bacterial cells, making it accessible for subsequent extraction and purification
steps, ultimately facilitating the isolation of high-quality microbial DNA for downstream analysis
and research applications.

B. PLANT DNA ISOLATION (CTAB METHOD)

I. CTAB
CTAB is used in plant DNA isolation, particularly in the CTAB method, because it helps
remove contaminants like plant cell polysaccharides and proteins. These contaminants can
interfere with DNA extraction and downstream applications. CTAB acts like a detergent,
breaking down cell walls and denaturing proteins, while also causing polysaccharides to
separate out. This leaves behind clean plant DNA that can be used for genetic research and
analysis.

II. NaCl
Sodium chloride (NaCl) is employed in the CTAB (cetyltrimethylammonium bromide)
method for plant DNA isolation to promote the separation of DNA from proteins and other
cellular components. In this method, NaCl is added to the plant cell lysate to create a high-salt
environment, which encourages the aggregation of proteins and cellular debris while DNA
remains in solution. This facilitates the subsequent precipitation of proteins, polysaccharides,
and other impurities when centrifuged, leaving behind purified plant DNA that can be further
processed and utilized for molecular biology applications, such as PCR and sequencing.

C. DNA ISOLATION FROM AN ONION

I. SDS

The reagent sodium dodecyl sulfate (SDS) aids in the disruption and solubilization of the
cell membrane, which is necessary for the separation of DNA. It accomplishes this by reacting
with the membrane's lipids, rupturing them, and releasing the cell's contents into the solution. By
destabilizing the tertiary and quaternary structures of proteins, SDS also plays a significant part
in denaturing (unfolding) proteins. As a result, the proteins can't function and can't obstruct the
DNA isolation procedure. SDS also aids in the DNA's own solubilization. The DNA strands are
surrounded and coated by SDS molecules, protecting their charges and enabling the DNA to
stay in solution. Proteins are kept in a denatured condition by SDS, which keeps them from
interfering with DNA precipitation or column chromatography or other separation processes.

II. Papain or Meat Tenderizer

In particular for enzymatic tissue digestion, papain and meat tenderizer are more
frequently used in the isolation of DNA from animal tissues. They have proteolytic enzymes that
can aid in the breakdown of animal tissues' proteins. Since the strong plant cell walls may be
broken down using techniques like CTAB or mechanical disruption, these enzymes are often not
needed when extracting plant DNA.

D. ANIMAL DNA
I. CHCL3: Isoamyl Alcohol
Animal DNA extraction requires the use of isoamyl alcohol and chloroform because they
allow for the separation, preservation, and purification of DNA. These compounds are crucial for
efficient DNA isolation in molecular biology research because they separate DNA from proteins
and cellular detritus, shield it from enzymatic breakdown, and allow for its retrieval as a pure
sample.

II. Isopropyl Alcohol

Isopropyl alcohol is used to precipitate DNA from an aqueous solution. When isopropyl
alcohol is added to a solution containing DNA, the DNA congeals and becomes visible as a
solid or gel-like substance. This makes the DNA visible, enabling its isolation.

2. Explain the principle involved in the following chemical tests for the
characterization of DNA:
A. Test for deoxyribose
The deoxyribose test works on the basis that deoxyribose sugar reacts with a particular
reagent to provide a visible response or color change. The Bial's Test is a typical test for
deoxyribose. Orcinol and concentrated sulfuric acid are both ingredients in Bial's reagent. In the
presence of sulfuric acid, deoxyribose and orcinol combine to produce a complex that, when
heated, glows green. Deoxyribose, a distinctive sugar present in DNA, is indicated by this color
shift.

B. Test for phosphate

The idea behind the phosphate in DNA test is that when phosphate ions interact with
particular chemicals, a precipitate is created. In the phosphate test, sulfuric acid and ammonium
molybdate are used to treat DNA. A yellow ammonium phosphomolybdate precipitate results
from this reaction. The existence of phosphate groups in DNA molecules is confirmed by the
creation of this yellow precipitate.

C. Test for purines

Purines in DNA, including adenine and guanine, may be detected using a test based on
how well they can absorb UV light. In the UV spectrum, purines exhibit distinctive absorption
peaks at particular wavelengths. When measuring the absorbance of DNA at 260 nm, where
purines largely contribute to the absorption, scientists frequently utilize a spectrophotometer.
This absorbance pattern aids in locating and measuring the purines in DNA.

D. Test for pyrimidines

Similar to the purine test, UV spectroscopy is used to detect pyrimidines in DNA.

Cytosine and thymine are pyrimidines with distinctive UV absorption patterns. At specified
wavelengths, they absorb UV radiation, with a peak absorption occurring at about 260 nm.
Based on the distinctive UV absorption spectra of pyrimidines, scientists may identify and
quantify the presence of pyrimidines in DNA by measuring the absorbance of DNA at 260 nm.

PAGE 89
ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS (RNA)

1. Identify the major types of RNA in a cell and discuss their individual functions in
relation to protein synthesis

The primary function of RNA is to create proteins via translation. Genetic information is
transported by RNA, and ribosomes transform it into a variety of proteins required for
cellular functions. Ribonucleic Acid (RNA) is specifically composed of three major types
involved in protein synthesis: Messenger RNA (mRNA), Transfer RNA (tRNA), and
Ribosomal RNA (rRNA).

A. mRNA
mRNA is produced during transcription from a DNA template. The function of mRNA is to
transport protein information from DNA in a cell's nucleus to the cytoplasm, or water-based
center, where the machinery responsible for producing proteins reads the mRNA sequence and
converts each three-base codon into an amino acid that belongs in a protein chain that is
expanding.

B. tRNA

The primary function of RNA is to create proteins via translation. Between the
messenger RNA (mRNA) molecule and the expanding chain of amino acids that make up a
protein, transfer RNA acts as a link (or adapter). A particular tRNA connects with its
complementary sequence on the mRNA molecule each time an amino acid is added to the
chain, ensuring that the right amino acid is put into the protein being made.

C. rRNA

The Ribonucleic acid (rRNA) is the RNA that may be found in ribosomes, which are the
molecules in charge of catalyzing protein synthesis. Ribosomal RNA makes up more than
60–80% of the ribosome's weight and is essential for all of the structure's functions, including
binding to mRNA, attracting tRNA, and catalyzing the synthesis of peptide bonds between
amino acids. A ribosome's three-dimensional structure is influenced by the three-dimensional
structure of a rRNA core. Ribosomal proteins contribute to the maintenance of this structure
through their interactions with the core.

2. Compare the structural differences of sugars and bases in DNA and RNA
molecules.

DNA is made up of two strands that are organized in a double helix. Nucleotides are the
building blocks that make up these strands. Each nucleotide has a phosphate, a nitrogenous
base, and a sugar molecule with five carbons. While RNA only has one strand, it is formed of
nucleotides, much like DNA. DNA strands are longer than those of RNA. RNA can occasionally
produce a secondary double helix structure.

3. What processing events differentiate eukaryotic mRNA from prokaryotic mRNA?

Prokaryotes produce a specific form of mRNA known as prokaryotic mRNA. Additionally,

it is polycistronic and consists of a number of structural genes from a specific operon.
Prokaryotic mRNA hardly ever changes post-transcriptionally. On the other hand, Eukaryotic
mRNA is the kind of mRNA found in eukaryotes and consists of the coding area of a single
gene. However, it goes through significant post-transcriptional changes such as splicing, the
inclusion of a 5′ cap and 3′ tail, and others. As a result, mRNA's structural and modificational
differences between prokaryotic and eukaryotic cells are the primary distinction.

PAGE 93 & 95
ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS (RNA)

1. Explain why the following reagents are used in the isolation of RNA:

A. Guanidine isothiocyanate

In RNA extractions, guanidinium thiocyanate is used to lyse cells and virus particles. In
addition to lysing, this compound denatures RNase enzymes to stop their activity. For RNA
extraction from entire tissue samples, particularly those that are high in RNase, guanidinium is a
reliable reagent. Nuclear RNA is released by the nuclear and organelle rupture caused by
plasma membrane solubilization, and they both copurify with cytoplasmic RNA species. The
presence of β-mercaptoethanol (β-ME) may improve the effectiveness of protein denaturation,
including the disruption of RNases.

B. β-mercaptoethanol

By breaking down disulfide bonds and removing the natural conformation needed for the
enzyme to operate, the reducing agent beta-mercaptoethanol (ß-ME) will irreversibly denature
RNases. Any RNases present in the substance to be extracted from will be totally inactivated
due to the powerful, but brief denaturing actions of the guanidinium isothiocyanate (GITC)
included in buffer RLT of the RNeasy Kits.

C. Phenol

Phenol is used in RNA isolation for its ability to denature and disrupt proteins, including
ribonucleases (RNases). RNases are enzymes that can degrade RNA, so it's essential to
inactivate them during the isolation process. Phenol helps inactivating RNases by denaturing
and precipitating proteins, effectively protecting the RNA. After mixing phenol with the
RNA-containing solution and centrifugation, the aqueous phase containing RNA can be
separated from the organic phase, which contains proteins and other contaminants.

D. CH3COONa

Sodium acetate is used primarily for RNA precipitation and purification. After the RNA is
extracted and treated with phenol, it is often necessary to remove any remaining contaminants
and concentrate the RNA. Sodium acetate is added to the RNA solution to create a high-salt
environment. This high-salt concentration promotes RNA precipitation by neutralizing the
charges on the RNA backbone, causing it to form a tight, insoluble structure. The precipitated
RNA can then be easily separated from the remaining solution through centrifugation, resulting
in purified RNA.

2. Why is RNA more susceptible to alkaline hydrolysis than DNA?

RNA is more susceptible to alkaline hydrolysis than DNA due to many structural
differences between these two forms of nucleic acids. DNA is double-stranded, whereas RNA
molecules are single-stranded. Since single-stranded nucleic acids lack a complementary
strand to protect them against enzymatic or chemical disintegration, they are typically more
vulnerable to hydrolysis. RNA contains ribose sugar, while DNA contains deoxyribose sugar.
The hydroxyl group (-OH) on the 2' carbon of ribose in RNA makes it more likely to undergo
hydrolysis in alkaline circumstances, which is the cause of this disparity. Saponification, also
known as alkaline hydrolysis, involves the dissolution of phosphodiester linkages in nucleic
acids. The RNA's 2'-hydroxyl group makes the neighboring phosphodiester bond more
susceptible to nucleophilic assaults from OH- ions, which finally causes the RNA backbone to
fracture.
 3. Provide the chemical nature or composition of the compound formed in a positive
result for the following:
A. Test for ribose

Positive Result: The Molisch Test can be used to confirm the presence of ribose.
Compound Formation: In a successful Molisch test, ribose reacts with concentrated sulfuric
acid to produce a complex combination. Typically, this complex combination contains furfural
derivatives, with furfural itself being one of the end products. The presence of carbohydrates is
confirmed by the appearance of a purple ring at the boundary between the acid and test layers.
If the test solution forms purple or purplish-red coloured layers.
B. Test for phosphate

Positive Result: The Ammonium Molybdate Test can be used to determine whether phosphate
ions (PO4-3-) are present.
Compound Formation: In this experiment, ammonium phosphomolybdate, a yellow precipitate,
is produced when phosphate ions interact with ammonium molybdate in an acidic solution.

C. Test for purines

Positive Result: The Xanthine Test, which requires the conversion of purines into xanthine and
subsequent reactions, can be used to identify the presence of purines.
Compound Formation: When the Xanthine Test is positive, xanthine is created when purines
are converted. The subsequent reactions of this xanthine can result in the production of uric
acid.

D. Test for pyrimidines

Positive Result: The Tolens' Test, which entails the conversion of pyrimidines into a chemical
known as Barbituric Acid, is used to identify pyrimidines.
Compound Formation: In a positive Tolens' Test, pyrimidines produce barbituric acid when
they react with nitric acid and heat. The distinguishing feature of this barbituric acid is its yellow
color, which is used to establish the presence of pyrimidines.