Dianne D.

Mariano
BSN 101

1. Define the following terms:

a. Nucleic acid - macromolecules made out of units called nucleotides, come in two naturally
occurring varieties: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is the carrier fo
genetic information.
b. Nucleotide - is the basic building block of nucleic acids. made up of three parts: a phosphate
group, a 5-carbon sugar, and a nitrogenous base.
c. Nucleoside - are the building blocks of DNA, consisting of a base (adenine, guanine, thymine,
cytosine, or uracil) linked to a sugar (ribose in RNA or deoxyribose in DNA).
d. Chromosome - are thread-like structures in which DNA is tightly packaged within the
nucleus. Chromosomes help ensure that DNA is replicated and distributed appropriately during
cell division. 
e. Gene - unit of hereditary information that occupies a fixed position (locus) on a chromosome.
Genes achieve their effects by directing the synthesis of proteins.
f. Genome - an organism’s complete set of DNA, including all of its genes. Each genome
contains all of the information needed to build and maintain that organism
g. Ribosome - is a complex made of protein and RNA and which adds up to numerous million
Daltons in size and assumes an important part in the course of decoding the genetic message
reserved in the genome into protein.
h. Transcriptome - is the set of all RNA molecules in one cell or a population of cells. It is
sometimes used to refer to all RNAs, or just mRNA, depending on the particular experiment.
i. Splicing - is a process that removes the intervening, non-coding sequences of genes (introns)
from pre-mRNA and joins the protein-coding sequences (exons) together in order to enable
translation of mRNA into a protein.
j. Exon - is any part of a gene that will encode a part of the final mature RNA produced by that
gene after introns have been removed by RNA splicing.
k. Intron - is any nucleotide sequence within a gene that is removed by RNA splicing during
maturation of the final RNA product. 
l. Alternative splicing - is a regulatory mechanism by which variations in the incorporation of
the exons, or coding regions, into mRNA leads to the production of more than one related
protein, or isoform.
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BSN 101

m. Codon - is a sequence of three DNA or RNA nucleotides that corresponds with a specific
amino acid or stop signal during protein synthesis.
n. Anticodon - are sequences of nucleotides that are complementary to codons. They are found
in tRNAs, and allow the tRNAs to bring the correct amino acid in line with an mRNA during
protein production.
o. Mutation - is a change that occurs in our DNA sequence, either due to mistakes when the
DNA is copied or as the result of environmental factors such as UV light and cigarette smoke. 
p. Mutagen - is a chemical or physical phenomenon, such as ionizing radiation, that promotes
errors in DNA replication.
q. Polymerase Chain Reaction - is a technique to make many copies of a specific DNA region
in vitro (in a test tube rather than an organism).
2. What is the difference between DNA and RNA?

The differences are as follows:

1. DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. The only
difference between ribose and deoxyribose is that ribose has one more -OH group than
deoxyribose, which has -H attached to the second (2') carbon in the ring.
2. DNA is a double-stranded molecule, while RNA is a single-stranded molecule.
3. DNA is stable under alkaline conditions, while RNA is not stable.
4. DNA and RNA perform different functions in humans. DNA is responsible for storing
and transferring genetic information, while RNA directly codes for amino acids and acts
as a messenger between DNA and ribosomes to make proteins.
5. DNA and RNA base pairing is slightly different since DNA uses the bases adenine,
thymine, cytosine, and guanine; RNA uses adenine, uracil, cytosine, and guanine. Uracil
differs from thymine in that it lacks a methyl group on its ring.

3. What are the components of a nucleotide?

Nucleotides are the building blocks of nucleic acids. They are composed of three subunit
molecules: a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one
phosphate group. Nitrogenous base - Purines or pyrimidines such as Adenine, Guanine, Cytosine,
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BSN 101

Thymine or Uracil. Sugar - Either ribose sugar or deoxyribose sugar. Phosphate group - 1 phosphorus
atom joined to 4 oxygen atoms

4. Describe the structure of a DNA double helix.

Double helix is the description of the structure of a DNA molecule. A DNA molecule consists of
two strands that wind around each other like a twisted ladder. Each strand has a backbone made
of alternating groups of sugar (deoxyribose) and phosphate groups. Attached to each sugar is one
of four bases: adenine (A), cytosine (C), guanine (G), or thymine (T). The two strands are held
together by bonds between the bases, adenine forming a base pair with thymine, and cytosine
forming a base pair with guanine.

5. Describe the base pairings found in DNA and RNA.

In DNA Adenine-Thymine and Guanine-Cytosine pair together due to the formation of hydrogen
bonds between the two bases. In RNA the base Thymine is not present, instead the base Uracil is
present which has a very similar structure to Thymine. As a result Adenine pairs with Uracil (A-
U) via the same hydrogen bonding interactions as in the A-T base pair.

6. Describe the processes involved in the replication of DNA molecules.

Preparation for Replication

Step 1: Replication Fork Formation

Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single
strands. DNA has four bases called adenine (A), thymine (T), cytosine (C) and guanine (G) that
form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds
with guanine. In order to unwind DNA, these interactions between base pairs must be broken.
This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen
bonding between base pairs to separate the strands into a Y shape known as the replication fork.
This area will be the template for replication to begin.

DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side
group is attached the DNA backbone. The 5' end has a phosphate (P) group attached, while the 3'
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BSN 101

end has a hydroxyl (OH) group attached. This directionality is important for replication as it only
progresses in the 5' to 3' direction. However, the replication fork is bi-directional; one strand is
oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging
strand). The two sides are therefore replicated with two different processes to accommodate the
directional difference.

Replication Begins 

Step 2: Primer Binding

The leading strand is the simplest to replicate. Once the DNA strands have been separated, a
short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as
the starting point for replication. Primers are generated by the enzyme DNA primase.

DNA Replication: Elongation

Step 3: Elongation
Enzymes known as DNA polymerases are responsible creating the new strand by a process
called elongation. There are five different known types of DNA polymerases
in bacteria and human cells. In bacteria such as E. coli, polymerase III is the main replication
enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA
polymerase III binds to the strand at the site of the primer and begins adding new base pairs
complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and
epsilon are the primary polymerases involved in DNA replication. Because replication proceeds
in the 5' to 3' direction on the leading strand, the newly formed strand is continuous.
The lagging strand begins replication by binding with multiple primers. Each primer is only
several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the
strand between primers. This process of replication is discontinuous as the newly created
fragments are disjointed.
Step 4: Termination
Once both the continuous and discontinuous strands are formed, an enzyme
called exonuclease removes all RNA primers from the original strands. These primers are then
replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to
check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki
fragments together forming a single unified strand. The ends of the linear DNA present a
problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the
parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective
caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of
DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the
ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils
into the familiar double helix shape. In the end, replication produces two DNA molecules, each
with one strand from the parent molecule and one new strand.
Replication Enzymes
DNA replication would not occur without enzymes that catalyze various steps in the process.
Enzymes that participate in the eukaryotic DNA replication process include:
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BSN 101

DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It
forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA
molecules that act as templates for the starting point of DNA replication.
DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and
lagging DNA strands.
Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from
becoming tangled or supercoiled.
Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain.
DNA ligase - joins DNA fragments together by forming phosphodiester bonds between
nucleotides.

7. What are the processes involved in the synthesis of protein?

Protein synthesis is the process in which cells make proteins. It occurs in two stages:
transcription and translation. Transcription is the transfer of genetic instructions in DNA to
mRNA in the nucleus. It includes three steps: initiation, elongation, and termination.

8. What are the types of RNA molecules?

In both prokaryotes and eukaryotes, there are three main types of RNA – messenger RNA
(mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

9. Describe the processes involved in transcription.

Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an
RNA molecule. RNA polymerase is the main transcription enzyme. Transcription begins when
RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through
helper proteins). RNA polymerase uses one of the DNA strands (the template strand) as a
template to make a new, complementary RNA molecule. Transcription ends in a process
called termination. Termination depends on sequences in the RNA, which signal that the
transcript is finished.

10. Describe the processes involved in translation.

Translation is a universal process in biology where a protein formed of amino acids is made by
using messenger Ribonucleic acid (mRNA) to dictate the order of amino acids.
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BSN 101

Following transcription (the conversion of DNA into mRNA) an mRNA strand leaves the

nucleus and enters the cytoplasm via the nuclear pore. Here it is recognised and becomes
attached to biological structures called ribosomes forming a 'polysome'. Each ribosome attached
to an mRNA strand will create a seperate polypeptide.
The bases (individual components of mRNA) are read in non-overlapping 3s (a codon) by the
ribosome. This is the 'triplet code'. Once mRNA has become attached the start codon is held in
the peptidyl (P) site and the next codon in the aminoacyl (A) site. There is also an exit (E) site
located on the other side of the P site. Synthesis occurs in a 5' to 3' direction of mRNA.
For amino acids to attach they are first conjugated (bonded) to a specific transfer RNA (tRNA),
each tRNA has an anticodon which is complementary to a codon in the mRNA. The tRNA and
mRNA are matched by codon to anticodon hydrogen bonding. This causes the conjugated amino
acid to be positioned so it can be bonded to the previous amino acid by a peptide bond. The start
codon, thus the first amino acid in a peptide is usually methionine.
The formation of the polysome and the attachment of methionine is known as the 'initiation' step.
Following initiation, the rest of the peptide is synthesised. The mRNA and tRNA-amino acid in
the A site are pushed into the P site, forcing that in the P site into the E site and a new codon
moves into the A site. At the E site the tRNA now without it's amino acid dissociates from the
mRNA and goes to bind another amino acid, it is recycled. This is called 'elongation' and it
repeats until a stop codon is reached. Causing the peptide chain to grow by one amino acid each
cycle.
Once a stop codon arrives at the A site there is no matching amino acid. Instead a release factor
protein triggers the disassembly of the ribosome-mRNA complex. This step is called
'termination'. Following this the protein is complete and is released by the ribosome.

11. What happens when a virus comes into contact with a host cell?
Viruses reproduce by infecting their host cells and reprogramming them to become virus-making
"factories."

12. What happens when a vaccine is injected into the body?

Your immune system reacts to the vaccine in a similar way that it would if it were being invaded
by the disease — by making antibodies. The antibodies destroy the vaccine germs just
as they would the disease germs — like a training exercise. Then they stay in your body,
giving you immunity.

13. Describe the processes involved in the production of Recombinant DNA.

1. Isolation of Genetic Material

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BSN 101

We already know that the genetic material of all living organisms is ‘nucleic acid’. In most
organisms, it is DNA, whereas in some it is RNA. The first step in rDNA technology is to isolate
the desired DNA in its pure form i.e. free from other macromolecules.

However, in a normal cell, the DNA not only exists within the cell membrane, but is also present
along with other macromolecules such as RNA, polysaccharides, proteins, and lipids. So, how do
we break open the cell and obtain DNA that is free from other macromolecules? We can use the
following enzymes for specific purposes:

 Lysozyme – to break bacterial cell wall.

 Cellulase – to break plant cell wall.

 Chitinase – to break fungal cell wall.

 Ribonuclease – removes RNA.

 Protease – removes proteins (such as histones that are associated with DNA).
Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of
ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified
DNA.

2. Restriction Enzyme Digestion

Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions
are called ‘restriction enzyme digestions’. They involve the incubation of the purified DNA with the
selected restriction enzyme, at conditions optimal for that specific enzyme.

The technique – ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction enzyme
digestion. This technique involves running out the DNA on an agarose gel. On the application of
current, the negatively charged DNA travels to the positive electrode and is separated out based on
size. This allows us to separate and cut out the digested DNA fragments. The vector DNA is also
processed using the same procedure.

3. Amplification Using PCR

Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence
using the enzyme – DNA polymerase. It helps to amplify a single copy or a few copies of DNA into
thousands to millions of copies. PCR reactions are run on ‘thermal cyclers’ using the following
components:

 Template – DNA to be amplified

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BSN 101

 Primers – small, chemically synthesized oligonucleotides that are complementary to a

region of the DNA.

 Enzyme – DNA polymerase

 Nucleotides – needed to extend the primers by the enzyme.

4. Ligation of DNA Molecules

The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us
the cut fragment of DNA and the cut vector, that is now open. The process of joining these two
pieces together using the enzyme ‘DNA ligase’ is ‘ligation’. The resulting DNA is ‘recombinant
DNA‘.

5. Insertion of Recombinant DNA Into Host

In this step, the recombinant DNA is introduced into a recipient host cell. This process
is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to
make them ‘competent’ to accept new DNA. (The topic – Tools of Biotechnology explains a few
ways to make cells competent).

During transformation, if a recombinant DNA bearing a gene for ampicillin resistance is transferred
into recipient E. coli cells, then the E. coli cells also become ampicillin-resistant. This aspect is
useful in differentiating transformed cells from non-transformed cells.

For example, if we spread the transformed cells on agar plates containing ampicillin, only the
transformed, ampicillin-resistant cells will grow while the untransformed cells will die. Therefore,
in this case, the ampicillin resistance gene acts as the ‘selectable marker’.

6. Obtaining Foreign Gene Product

The recombinant DNA multiplies in the host and is expressed as a protein, under optimal
conditions. This is now a recombinant protein. Small volumes of cell cultures will not yield a large
amount of recombinant protein. Therefore, large-scale production is necessary to generate products
that benefit humans. For this purpose, vessels called bioreactors are used.

Bioreactors are large containers with a continuous culture system, where the fresh medium is added
from one side and used medium is taken out from another side. Bioreactors can process about 100-
1000 litres of cell cultures. A bioreactor provides optimum conditions (temperature, oxygen, pH,
vitamins etc.) to biologically convert raw materials into specific proteins, enzymes etc.

‘Stirred-tank bioreactor’ is the most common type of bioreactor. It is usually cylindrical and has the
following parts:
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BSN 101

 Agitator system – to stir the contents evenly

 Oxygen delivery system – to introduce air into the system

 Foam control system

 Temperature control system

 pH control system

 Sampling ports – to take out small amounts of culture

7. Downstream Processing

Before the protein is marketed as a final product, it is subjected to downstream processing which
includes:

 Separation and purification.

 Formulation with suitable preservatives.

 Clinical trials to test the efficacy and safety of the product.

 Quality control tests.