MODX 311: Molecular Biology and Diagnostics│Lecture

2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

1949 SICKLE CELL ANEMIA MUTATION

Nucleic Acid and Protein WAS FIRST STUDIED
Structure and Function 1953 WATSON AND CRICK’S DNA
(Preliminary Term, 2nd Topic) STRUCTURE
Trans Outline: 1970 RECOMBINANT DNA TECHNOLOGY
I: History of Molecular IV: Ribonucleic acid (RNA) 1977 DNA SEQUENCING
Diagnostics a. Types of RNA 1985 IN VITRO AMPLIFICATION OF DNA
II: Nucleic Acid 1. Ribosomal RNA (RRNA) (PCR)
a. Types 2. Messenger RNA (MRNA) 2001 THE HUMAN GENOME PROJECT
- Deoxyribonucleic acid 3. mRNA Transcription
(DNA) 4. Transfer RNA (tRNA)
- Ribonucleic acid (RNA) 5. Small Nuclear RNA
b. Structure (snRNA) VERY IMPORTANT: 1953
III: Deoxyribonucleic acid 6. Other RNAs o 1953 – It is one of the most important biological
(DNA) b. Transcription discoveries of the 20th century when James Watson and
a. DNA structure 1. Transcription Initiation
b. DNA Double Helix 2. Transcription Francis Crick described the structure of DNA.
Comprehension Check ** Elongation o 1865 – Gregor Mendel discovered that the traits of
c. DNA Replication 3. Transcription
parents could be passed through to the children.
1. DNA polymerase Termination
d. Restriction Enzymes V: Other RNA-Metabolizing o 1985 – Kary Mullis discovered the in vitro
1. DNA Degradation Enzymes amplification of DNA.
2. Three types of Retriction 1. Ribonucleases
Endonucleases 2. RNA Helicases ▪ He won a noble prize because of the discovery
-TYPE 1 VI: Proteins of the Polymerase Chain Reaction (PCR).
-TYPE 2 VII: Amino acids o 2001: They tried to know the sequence of entire gene
-TYPE 3 a. Primary Structure
e. Recombination (Sexual b. Secondary Structure of a human starting from the chromosome #1 up to the
Reproduction) c. Tertiary Structure sex chromosomes.
f. Recombination (Asexual d. Quaternary Structure
Reproduction) VIII: Chromosomes
g. Plasmids a. Chromosome Formation NUCLEIC ACID
VIX: Definition of Terms o The building blocks of DNA and RNA are made up of
a. Karyotype
b. Genotype
nucleic acids.
c. Phenotype o One of the macromolecules in our body together with
the carbohydrates, lipids, and proteins.
o Nucleic acid: polymers of nucleotide.
HISTORY OF MOLECULAR DIAGNOSTICS • Macromolecules are constructed out of long chains
• It was used to describe the study of DNA, RNA and (strands) of monomers called nucleotides.
proteins. o Each nucleotide is made up of nitrogenous bases,
• In molecular lab, the techniques are designed for sugar and phosphate groups.
handling and at the same time you want to analyze Each nucleotides have three functional groups:
nucleic acids (DNA/RNA). 1. Nitrogenous base
• For us to know the sequence of nucleotides that makes 2. Pentose Sugar
up the nucleic acid, that serves as basis of normal and
3. Phosphate groups
pathological traits from microorganisms.
• The main function of the Nucleic acid is to store and
• This provides a powerful means of predictive analysis.
transmit the genetic information from the DNA to
• As a MedTech, be knowledgeable of the structure and
become protein.
function of nucleic acids.
o They have structural and catalytic components.
YEAR KEY EVENT • DNA is very important for the storage of genetic
1865 MENDEL’S LAW OF HEREDITY information of the living organism.
1866 JOHANN MIESCHER, PURIFICATION
OF DNA

1
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
TWO TYPES:
• Deoxyribonucleic acid (DNA): serves as genetic
material of all genetic organism.
• Ribonucleic acid (RNA): carries information but
most of the time, they transmit information coming
from DNA to have a product.
o Information from the DNA will be transcribed to
become RNA and later be translated to protein.
o Translates the genetic material (DNA) to become
a phenotype.
o VIRUSES: also carries genetic material; uses
RNA as their genetic code.
STRUCTURE
• Nitrogenous bases:
o To differentiate DNA and RNA, check the
pyrimidine bases (T vs U) and the sugar building
block (Deoxyribose vs Ribose).
❖ Guanine
❖ Adenine
❖ Cytosine
❖ Thymine – specific for DNA
❖ Uracil – specific for RNA
o 2 types of Nitrogenous bases: Purine and Pyrimidine
bases.
o PURINE – made up of two or double ring structure
(Adenine and Guanine). *PURAG2
o PYRIMIDINE: has single ring structure (Thymine
and Cytosine). *PYTC1
o Sugar building block of the Nucleic acid – Pentose
sugar.
- These sugars have five carbons. Each carbon has
their own function. Based on the hydroxyl group,
differentiate if ribose or deoxyribose on 2nd
carbon.
1st carbon holds the nitrogenous bases
2nd carbon determines whether it is Deoxyribose or
Ribose
-OH (Hydroxyl group) – ribose; Oxygenated
hydrogen
-H (Hydrogen group) – Deoxyribose; Deoxygenated
(no presence of O2)
3rd carbon: holds -OH group or hydroxyl that
attaches to the phosphate group of the succeeding
nucleotides forming a sequence of nucleotides.
5th carbon: holds the phosphate group and binds to
the hydrogen group of the 3’ end of the succeeding
nucleotides through phosphodiester bond. It also
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
supports the sugar phosphate backbone, that is made
up of phosphate and a sugar.
Sugar Carbons are 1-5: 1st nitrogen base, 2nd
hydroxyl, 3rd is for phosphodiester bonds
3rd and 5th Carbon are used to form the long strand of
DNA.
For example. There are 2 bases, 5’ Carbon is where
phosphate group is located. 3’ Carbon is where the
Hydrogen group is found.
o Each strand of DNA or RNA has 5’ end and 3’ end.
o 5’ end: ends with free phosphate group (no sugar
attached).
o 3’ end: ends with free sugar (no phosphate group
attached).
o Nucleotide = Phosphorylated sugar + Base
o Nucleoside= Sugar + Base w/o the phosphate group
o Not all DNA are double stranded, and not all RNA is
single stranded.
DEOXYRIBONULEIC ACID (DNA)
• Primary function is to store genetic information.
o Usually found in nucleus, and some (small
amount) are found in the mitochondria.
o Macromolecule of phosphorous, carbon,
hydrogen, oxygen, and nitrogen (PCHON)
atoms.
o Assembled in units of nucleotides that are
composed of a phosphorylated deoxyribose
sugar and a nitrogen base.
o Nitrogen bases are attached to the deoxyribose
sugar which forms a polymer with the
deoxyribose sugars of the other nucleotide
through a phosphodiester bond.
o Partners for Life= (A & T) and (C & G)
- The linear assembly of nucleotide makes up 1
strand of DNA. 2 strands of DNA comprise the
DNA double helix.
DNA STRUCTURE
• Double helical structure
• First Described by James Watson and Francis
Crick
o Their molecular model of DNA was founded on
previous observation of the chemical nature of
DNA including the Diffraction analysis
performed by Rosalind Franklin.
2
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
▪ There was a debate who discovered the
structure of DNA.
❖ Watson & Crick: formed the model and
thus was credited for the structure.
❖ Franklin: performed diffraction
analysis where it became the basis of
Watson and Crick’s study.
• Helical structure of DNA results from specific
sequence (order) of nucleotides in the strand, as well
as the surrounding chemical microenvironment.
• Two DNA chains form hydrogen bonds with each
other in a specific weight
o These hydrogen bonds between the nucleotide are
the key to the specificity of most nucleic acid base
test in Molecular Laboratory.
o The specific hydrogen bond formation is how the
information is held in linear order of the
nucleotides that is why the double stranded
structure is being maintained.
o Hydrogen bond should be complementary.
*Complementary Nucleotide: A must be paired with T.
Cytosine must be paired with G. In this way, the parental
DNA strand can be replicated, without loss of nucleotide
order.
*Base pairing: Ex. when A bound to C, it results to
mismatches. It can distort DNA Helix which could disrupt
maintenance of sequence information and must be removed
before translation through RNA splicing.
*(Before going to the ribosome for translation, removal of
non-complementary/ unwanted strand is done).
*Also, during the DNA replication, there are enzymes
(exonuclease enzymes) that destroys non-complementary
bases to maintain the sequence information.
DNA DOUBLE HELIX
• The sequences of the two strands that form the double
helix are complementary.
• Base pairing which follows Chargaff’s rule
(complementary pairing)
o The Adenine should bind with Thymine, and Cytosine
should bind with Guanine.
o Binding of 2 purine or 2 pyrimidine together is not
possible because:
o The bounded pair will sort of fit to diameter of
double.
o If one is double ring structure, the other one must
be single ring to maintain the size of double helix
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
because that is the only arrangement that can fit
the structure.
o There is no two double ring structure that binds
together. It is not possible because these bonded
pairs will sort of fit in the diameter of the double
helix.
o If you will bind two purines, the diameter of the
DNA will be increased.
o It should be 1 Purine/1 double ring structure and
1 single ring structure.
o The backbone of the DNA is made up of sugar-
phosphate backbone (important for the helix
formation).
o For the base pairing, nitrogenous bases have a certain
pattern.
- A-T= 2 H bonds
- C-G= 3 H bonds
o Between the two, the Guanine and Cytosine are more
stable compared to the Adenine and Thymine which
are easy to be denatured.
• The complementary strands are Antiparallel
orientation, meaning the 5’ end of one strand binds
with the 3’ end of the other strand. That is how the
DNA is being replicated.
o Once the base pair interaction forms the ladder. DNA
ladder/Structure will be twisted/coiled. It forms as a
double helix because of nitrogenous bases. DNA is
twisted because:
- Nitrogenous bases are hydrophobic. Sugar will
twist to prevent the water to come in contact with
the nitrogenous bases.
- Unlike the Sugar phosphate backbone, they are
Hydrophilic (water-loving) in nature.
*The nitrogenous bases are oriented towards center. Outer
part of DNA is the backbone (sugar and phosphate group).
They are highly specific and they held together in a certain
pattern.
● The formation of hydrogen bonds between two
complementary strands of DNA is called
hybridization.
COMPREHENSION CHECK
• Anti-parallel, double stranded molecule
• Sugar Phosphate backbone
• Complementary base pairs joined by Hydrogen
bond in the middle.
• Each strand has the potential to deliver and code for
information.
3
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
• Length of DNA given in base pairs (1,500 base pairs
of nitrogenous bases)
• ALWAYS READ THE STRAND FROM 5’ TO 3’.
o 5’ is the first one to be synthesized during the
elongation step of the nucleic acids.
o 3’ is continuously elongated.
DNA REPLICATION
*It is semi-conservative because the DNA replication
results to two stranded-DNA (one is conserved, one is
new).
*Semi-conservative replication is the key to maintaining
the sequence of the nucleotides in DNA through new
generations. It is important that this information, in the
form of the DNA sequence, be transferred faithfully at cell
division.
*The replication apparatus is designed to copy the DNA
strands in an orderly way with minimal errors before each
cell division.
*Parent strand will be unwind/ unzipped which serves as
template to form a new strand of DNA. 2 strands of DNA
have antiparallel orientation because of the way the DNA
is replicated.
1. DNA polymerase
• Enzyme responsible for polymerizing the nucleotide
chains.
o Polymerization: Elongates the nucleotide chain.
o Responsible to add more nucleotide bases is DNA
polymerase by using a guide or template.
• Template which came from the parent strand is
used to determine which nucleotides to add to the
chain.
o Resulting strand is made up of the parent strand
which serves as the template and the newly
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
synthesized strand (blue part of the helix from the
picture).
o DNA synthesis proceeds from 5’ to 3’ direction
but the part where DNA polymerase will attach is
on the 3’ end because this is where
polymerization continuously happens (there is no
addition of nucleotides on the 5’ end).
o It reads the template in the 3ʹ to 5ʹ direction.
*Other key players for the DNA Replication:
o They are made up of enzymes except the single
stranded binding proteins.
o It actually starts as a double stranded DNA, but
we have to unzip the DNA strand.
✓ HELICASE – This will unzip the DNA strand. The
site where the strand is being unwound by the helicase
enzyme is called replication fork.
o Usually, before the replication fork, we have enzymes
present here, which is the topoisomerase.
✓ TOPOISOMERASE – placed in front of the
replication fork. This enzyme will prevent the super
coiling of the DNA during the unzipping of the
complementary strand.
✓ SINGLE STRANDED BINDING PROTEINS –
This will bind to the template/ parent strand to prevent
the re-binding/ re-hybridization of the
complementary bases.
o It is used to keep each strand apart. Because when they
have already separated, there is a tendency to rebind.
✓ RNA PRIMASE – It will prime the synthesis of new
strand of DNA. Found in 3’ end of the parent
strand. It very important to produce short RNA
primers (6 to 11 bp) which is used for priming DNA
synthesis. An RNA primer is a short strand of
nucleotide that are specific/ complementary to the
parent strand. They will dictate where to start the
replication. Primase must work repeatedly on the
lagging strand to prime synthesis of each Okazaki
fragment.
o For example: the parent strand (yellow strand in the
picture): the priming starts on its 3’ end. It is the
starting point of the synthesis. It is where the new
strand of DNA begins.
*RNA primase is the one who binds and sends signals to
the DNA polymerase III, so the DNA polymerase III will
now find the part where there are RNA primers; so, from
the RNA primers it will start the synthesis. (The primer is
the blue strand in the picture, the 3’ and 5’ end in the
parent strand where the primers produce the 5’ end) it
will be copied towards the 5’ end of the parent cell.
4
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
So, where will the DNA polymerase bind?
o At the 3’ end of the RNA primer to form or to
synthesize a new strand of it. The replication of the
DNA polymerase is from 3’ end of the parent side
towards the 5’ end; it can be noticed that the formation
goes to the end of the 5’ end of the parent strand.
o Look at the strand that being build (new daughter
strand in the picture) remember our RNA will always
build from 5’, where the primer and 3’ will start so
that it could continuously add some bases.
✓ These small fragments, or Okazaki fragments, were
the key to explaining how both strands were copied at
the replication fork. The two strands of the parent helix
are not copied in the same way. While DNA
replication proceeds in a continuous manner on the
3′ to 5′ strand, or the leading strand, the replication
apparatus jumps ahead a short distance (~1,000 bases)
on the 5 ′ to 3 ′ strand and then copies backward
toward the replication fork. The 5 ′ to 3 ′ strand
copied in a discontinuous manner is the lagging
strand.
*The top strand (leading strand in the picture), the lower
strand (lagging strand in the picture).
*The leading strand would form a new strand towards the
replication fork, whereas a lagging strand, it forms a new
strand away from the replication fork and just fragments
(Okazaki fragment in the picture).
Why are only fragments one that formed?
o It is because the RNA primer would bind and will form
a short strand; once a fragment is completed by the
DNA polymerase, it will come off and removed to those
primers. What will happen is that the primer would
bind and form a strand then the DNA polymerase I
would remove it and the RNA primers will bind again
to another part of the DNA, would form another
strand, and the DNA polymerase I would remove it
again and continuously do it. That is why there are
lags and only primers and not a continuous formation
of strands, the position of primers is lagging.
o DNA polymerase I is the one that removes the
primers, but the one that catalyzes the addition of
nucleotides to the growing strand of the DNA is DNA
polymerase III.
So, how will the lagging strands be connected?
o Okazaki fragments can form continuous strand
through the enzyme DNA ligase, the ligase enzyme is
responsible for connecting the fragments since the
DNA polymerase I removes the RNA primers
resulting to lagging fragments.
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
o DNA polymerase I also known as Rnase H (removes
the RNA primers from the lagging strand, only in
lagging strand).
o DNA polymerase III catalyzes the addition of new
nucleotides baes to form the new strand of the DNA.
o This all happens in vivo, inside the cell, before mitosis
replicate the DNA.
RESTRICTION ENZYMES
*They cut the DNA (nucleases, DNases are responsible for
DNA degradation).
DNA DEGRADATION
1. EXONUCLEASES
o One purpose of the exonuclease function in the
various DNA polymerases is to protect the
sequence of nucleotides, which must be faithfully
copied.
o Copying errors will result in base changes or
mutations in the DNA. The 3 ′ to 5 ′ exonuclease
function is required to ensure that replication
begins or continues with a correctly base-paired
nucleotide.
o The enzyme will remove a mismatch (e.g., A
opposite C instead of T on the template) in the
primer sequence before beginning
polymerization. During DNA synthesis, this
exonuclease function gives the enzyme the
capacity to proofread newly synthesized DNA,
that is, to remove a misincorporated nucleotide by
breaking the phosphodiester bond and replace it
with the correct one.
o It cuts or degrades the DNA on one end, from the
end of the DNA strand.
2. ENDONUCLEASES
o They degrade usually in the middle portion.
• Restriction enzymes (molecular scissors)
• Attacks specific sequence of DNA and break or
restrict the DNA polymer at the sugar-phosphate
backbone.
o They can cut in the middle to form 2 separate
DNA strand.
o They are originally produced by bacteria
(bacteriophages) but later on they discovered
that even human cells and all nucleated cells has
endonucleases.
5
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
THREE TYPES OF RESTRICTION
ENDONUCLEASES
• TYPE 1
o Random cuts are made, anywhere in the DNA
o have both nuclease and methylase activity in a
single enzyme
• TYPE 2
o Mostly used in the laboratory, specific cut, most
useful if you want to target specific sequence of
DNA.
o These enzymes do not have inherent methylation
activity.
o Type II restriction enzymes recognize
symmetrical DNA sequences and cut the sugar-
phosphate backbone in different ways, leaving no
single strands at the cut site (blunt ends) or 5 ′ or
3 ′ overhanging single-stranded ends. Exposed
single-stranded ends are “sticky” ends that can
hybridize with complementary overhangs.
o For example, the restriction area EcoRI from E.
coli R1 strain; what happen here is that
restriction enzyme area EcoR1 will find target
sequence (refer to the picture the GAATTC) once
it has found that sequence, it will cut in between
adenine and guanine; that is what you called a
restriction of enzyme digestion.
• TYPE 3
o Non-specific cuts
o Although not completely characterized, type III
restriction enzymes resemble type I enzymes in
their ability to both methylate and restrict (cut)
DNA. Like type I, they have multiple subunits,
including helicase (unwinding) activity.
Recognition sites for these enzymes are
asymmetrical.
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
o All of them originated from organisms and they have
specific recognitions sites
• Deoxyriboendonucleases or Endonucleases
- Break the sugar-phosphate backbone of DNA.
o They cut the bond between the bases; the
difference between endo (middle) and exo (end
portion; usually at 3’ is the location where they
cut.
• Restriction enzymes
- Endonucleases that recognize specific base
sequences and break or restrict the DNA polymer
at the sugar-phosphate backbone
o These are endonucleases that we are using in the
lab as a molecular scissors; if you want to cut the
DNA on the specific target sites then you can use
restriction enzymes.
• DNA Ligase
- Catalyzes the formation of a phosphodiester bond
between adjacent 3ʹ-hydroxyl and 5ʹ-phosphoryl
nucleotide ends
o To connect or form a single continuous
strand of DNA; since the lagging strand has
okazaki fragments and we have to connect
the okazaki fragments into a very long
continuous strand of DNA.
• Other DNA Metabolizing Enzymes :
✓ Nucleases- degrade DNA from free 3ʹ-hydroxyl or 5ʹ-
phosphate ends.
✓ Methyltransferases- catalyze the addition of methyl
groups to nitrogen bases
✓ Helicases- separation of the sugar-phosphate
backbones in both strands
- Use to unzip or unwind the double stranded DNA
to become a single stranded DNA
6
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

RECOMBINATION (SEXUAL REPRODUCTION) o For example, bacteria is infected with bacterial

• mixture and assembly of new genetic combinations
o Very good example for this one is the genes coming
from our parents, usually actually combination of our
parents gene and we are the new genetic combination.
o It occurs through molecular process; there is a
crossing over or physical exchange between molecules
o Recombinant molecules is the one that holds a new
combination of DNA sequence; your DNA sequence is
different from your mother or your father because you
are the combination of both.
o Based on the Mendel’s law, each generation of
sexually reproducing organism is a new combination
of the parental genomes; so yung parent genes will be
mixed to form the child’s gene.
3. Transformation
RECOMBINATION (ASEXUAL REPRODUCTION)
viruses or bacteriophage, which are intracellular
parasites (uses the capability ng cell to replicate
nucleic acid); the virus will join with the nucleic
acid of the organism and it will produce a new
strand or new recombination and that could infect
another cell, so the combination of the new strand
on the bacteria and the virus that will carry the
hereditary strand from one bacterium to another
is called the transduction.
o These viruses to insert their DNA through the cell
wall into the bacterial cell. The DNA of a bacterial
virus was the carrier of its genetic determination
in the transduction process. The viral protein
remained outside of the cell, whereas viral DNA
entered the cell.
o Although conjugation and transduction were the
methods for the initial study of the connection
between DNA and phenotype, transformation, is
the basis for modern-day recombinant techniques.
o This is like an in vitro situation or is induced by
recombination; you can do genetic engineering or
recombination in vitro.
o Today, nucleic acids or DNA try to alter the DNA
of certain organisms to check if there is any
changes or functions; for some laboratories they
do some experimentation or transformation of
certain organism

PLASMIDS
*Mostly seen in bacteria.
*Do bacteria have a linear genetic material?
o No, they are circular in form.
• Genetic information in asexually reproducing
o Plasmids are mostly circular form in contrast to human
organisms can be recombined in three ways:
chromosomes and other organisms such as fungi,
1. Conjugation
plants, and animals that are moslty linear but in
o We can transfer a genetic material to another
bacteria they are circular; DNA could be linear or
through direct contact.
circular in form.
o Asexual reproduction often occurs in bacteria.
o Bacteria can own a chromosome complement
Example, there is a donor bacteria for the genetic
(plasmids).
material and recipient bacteria (refer to the 2nd
o Most plasmids are double-stranded circles of 2,000
diagram), so the donor bacteria carries the DNA
to 100,000 bp (2 to 100 kilobase pairs) in size.
sequence called the fertility factor or the F –
o Plasmids can carry genetic information.
factor (the one that is eclosed to red circle in the
o They can carry genetic information but they can
picture). There will be recombination now.
only carry limited amount of information and
2. Transduction
usually the resistant gene of bacteria
o transmission of hereditary units carried by viruses
from one bacterium to another .

7
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

• Plasmids were found to be a source of resistant

phenotypes in multidrug- resistant bacteria.
o Multi resistant bacteria, resistant phenotypes of
MDRO’s (multidrug-resistant organisms) by having
specific genes present in their plasmids

TYPES OF RNA
A. RIBONUCLEIC ACID (RNA) RIBOSOMAL RNA (RRNA)
o Most of the RNA are single-stranded (some are
• The largest component of cellular RNA
double-stranded, that is why you cannot differentiate
• 80% to 90% of the total cellular RNA (because
DNA and RNA based on the number of strands).
ribosomal RNA is where the translation happens)
o The RNA translate the DNA sequence into proteins.
• Important structural and functional part of the
o Most of the RNA are found in the cytoplasm.
ribosomes, cellular organelles where proteins are
o RNA can also be found in the nucleus, which are the
synthesized
mRNA (type of RNA that did not leave the nucleus).
• Various types of ribosomal RNAs are named for their
Can we isolate RNA from the nucleus?
sedimentation coefficient (S) in density-gradient
o They are much smaller from DNA
centrifugation.
• Polymer of nucleotides similar to DNA
• Three rRNA species in prokaryotes:
• Synthesized as a single strand rather than as a double
▪ 16S, 23S, 5S
helix
o There is small and large subunit in the ribosome
• RNA strands do not have complementary partner
which is used in translation (where the mRNA
strand
passes during the translation)
• Nitrogen bases
o The 16S found in the ribosome small subunit
▪ Adenine
and the 23S and 5S found in the ribosome large
▪ Cytosine
subunit, all synthesized from the same gene.
▪ Guanine
• rRNA species in Eukaryotes
▪ Uracil
▪ Copied from DNA as a single 45S precursor
RNA (pre- ribosomal RNA)
o Another rRNA species, 5S, found in the large
ribosome subunit in eukaryotes, is synthesized
separately.

MESSENGER RNA (MRNA)

o Messenger RNA (mRNA) is the initial connection
between the information stored in DNA and the
translation apparatus that will ultimately produce the
protein products responsible for the phenotype.
o Transports information from DNA to ribosomes
• In prokaryotes, mRNA is synthesized and
simultaneously translated into protein.

8
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
o
The information from the DNA is genotype;will
become a phenotype or the feasible characteristic
of the organism if it become a protein.
○ Genotype - information in the DNA
○ Phenotype - when it becomes protein. the
visible characteristic or organism.
● Prokaryotic mRNA is sometimes polycistronic
○ Polycistronic - One mRNA can code multiple
proteins.
● Eukaryotic mRNA is monocistronic,
○ Monocistronic - A single mRNA will only code for
a single protein only (the gene in human are very
specific).
○ Example: I have a gene, the NADPH. It will only
produce the NADPH structure, it will not create
another type of molecule/ substance.
○ Most of the eukaryotic mRNA are monocistronic
o In eukaryotes, copying of RNA from DNA and
protein synthesis from the RNA are separated by
the nuclear membrane barrier.
o Eukaryotic mRNA undergoes a series of post-
transcriptional processing events before it is
translated into protein.
mRNA Transcription
Two types of transcription:
● Constitutive Transcription
o Some of the messages are transcribed constantly.
It is constant because even when it is not needed,
it is still being produced by the cell.
o That’s why when you check their presence in the
cell, they are relatively abundant.
o Constitutive = constant in transcribing and
translating.
● Inducible or Regulatory Transcription
o Transcribe only certain times during cell cycle or
under particular conditions.
o It will not be produced, unless it is induced.
o Not constant, should be induced.
o Should be induced by a certain factor before it
will be transcribed.
TRANSFER RNA (tRNA)
● Responsible for carrying amino acids to the
ribosome.
○ The amino acid they carry is a triplet of amino
acid, which is called Codon.
○ There’s codon and anticodon.
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
○ Codon: mRNA, Anticodon: tRNA
○ tRNA (that carries amino acid; where translation
happens). mRNA (that carries the genetic
material coming from the DNA)
● Translation of information from nucleic acid to
protein requires reading of the mRNA by
ribosomes, using adaptor molecules or transfer
RNA (tRNA)
● Relatively short, single-stranded polynucleotides of
73 to 93 bases in length
● MW 24,000 to 31,000
SMALL NUCLEAR RNA (snRNA)
● Functions in splicing in eukaryotes
● snRNA stays in the nucleus after its transcription by
RNA polymerase I or III.
○ RNA splicing - is used to remove non-
complementary bases before translation
occurs.
○ After transcription, it will undergo RNA
splicing on the nucleus before going to the
ribosome for translation.
● RNAs sediment in a range of 6 to 8S
OTHER RNAs
● Untranslated RNA
- sRNAs (bacteria)
- ncRNAs (noncoding RNA; eukaryotes)
● They don’t have much effect when it comes to
transcription and translation.
Gene expression - undergoes transcription-translation. It
is called as gene expression because we have expressed the
information from genome to become protein. From
genotypic, it would be phenotypic.
TRANSCRIPTION
• Happens within the nucleus (the DNA that carry the
information)
○ In order for the information to be utilized, it must
be transcribed and then later translated into
protein.
● Copying one strand of DNA into RNA by a process
similar to that of DNA replication.
● Catalyzed by RNA polymerase, occurs mostly in
interphase
● Three Types of RNA polymerase
○ pol I - used for non-coding RNA
9
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
○ pol II - used for coding RNA, responsible for
the synthesis of mRNA (carries genetic
information to be translated into protein).
○ pol III - used for non-coding RNA
● How transcription happens?
○ We have to ensure that we unwind the part
that will be transcribed. Entire DNA is not
unwound, only the specific part of the gene or
the genome.
○ We have to unwind it first through the use of
helicase enzyme.
○ What will happen is that only 10 base pairs of
DNAs will be opened to allow RNA
polymerase to work. Only short part will be
opened.
1. TRANSCRIPTION INITIATION - during
transcription initiation, RNA polymerase will
assemble to the specific site of DNA called the
promoter.
2. TRANSCRIPTION ELONGATION - RNA
polymerase (both prokaryotes and eukaryotes) will
now synthesize RNA using the base sequence of one
strand of double helix as its guide. RNA polymerase
will prime the synthesis of the new strand of mRNA,
it will be elongated and for the copy of guide or the
template.
o The complementary strand of the DNA template
(that is not copied) has a sequence identical to
that of the RNA product (except for the U for T
substitution in RNA).
o Remember: Only one template will serve as a
guide. It uses one-strand of the double helix and
usually, it’s the antisense strand that will serve
as a guide. Another strand would not be copied.
3. TRANSCRIPTION TERMINATION- happens
when RNA synthesis that is responsive to the
protein products will stop. It will induce the
termination of synthesis of mRNA.
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
o If you are forming too long strand, it should be stopped
by the transcription termination.
Remember: Only type II is used for mRNA.
OTHER RNA-METABOLIZING ENZYMES
Ribonucleases
● Degrade RNA in a manner similar to the degradation
of DNA by deoxyribonucleases
○ Classification:
■ Endoribonucleases - inside the RNA.
■ Exoribonucleases - used to cut unwanted
or non-complementary bases.
RNA Helicases
● Catalyze the unwinding of double-stranded RNA
● Important to unwind the specific parts of DNA where
transcription will occur or takes place.
PROTEINS
• Most abundant when it comes to other
macromolecules in our body because most of the
information in our gene will be later on translated to
become proteins or to become the phenotypic type.
○ Proteins are the product of molecular synthesis
of DNA and RNA. Later on, because of
transcription and translation, they will be
produced.
● Products of transcription and translation of the
nucleic acids.
● They manifest the phenotype directed by the nucleic
acid information.
● Polymers of amino acids
○ In order to interpret the nucleic acid analysis
result accurately, it is important to understand
the movement of genetic information from DNA
to protein as dictated by the genetic code.
● Proteins are polypeptides that can reach sizes of
more than a thousand amino acids in length
10
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

○ the information stored in the DNA is backbone which is the Carbon-Carbon-

translated into amino acid Nitrogen.
● Most abundant macromolecule in cells
● Proteome vs Genome
○ Proteome - collection of proteins
encoded in all organisms
○ Genome - collection of genes
AMINO ACIDS
• Amino acids are the building block of proteins
• Composed of four different groups:
1. Carboxyl Group
2. Amino Group
3. Side chain Group
4. Carbon Group (attached to these three
distinct groups which are the carboxyl, side
chain, and amino group)
• When it comes to the activity of the amino acid, they
are at pH 7 and they become ionized.
• Most of the carboxyl groups of amino acids are ionized
at pH 7 and the amino groups are not.
• At physiological pH, the charge of amino acids is a
negative charge
• Amino acids are zwitterions at physiological pH
• At certain pH level, amino acids can be completely
positive or negatively charge.
○ At pH 7, most of the carboxyl groups of the amino
acids are ionized, and the amino groups are not. The
ionization can switch between the amino and carboxyl
groups, making the amino acids zwitterions at
physiological pH. At certain pH levels, amino acids
will become completely positively or negatively
charged.
● Each amino acid has characteristic biochemical
properties determined by the nature of its amino acid
side chain
● Grouped according to their polarity (tendency to
interact with water at pH 7) as follows: nonpolar,
uncharged polar, negatively charged polar, and
positively charged polar
● The properties of amino acids that make up a protein
determines the shape and biochemical nature of the
protein.
○ There is an amino acid sequence because there is
a joining of the amino group and the carboxyl-
terminal group of the amino acids so there is a
binding. The linkage or bond in between the
amino acids to form the protein backbone is the
peptide bond. Peptide bonds formed this protein

11
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
PRIMARY STRUCTURE
● The linear sequence of amino acids joined together by
peptide bonds
○ Primary structure is a very long sequence of
amino acids
○ The sequence of amino acids in a protein
determines the nature of the activity of the protein
○ This sequence is read by:
■ Start to the amino-terminal and it will
end with a carboxyl-terminal end.
○ The backbone in the middle is the C-C-N group
that is bound to peptide bond (in between the
amino group and the carboxyl group of the other
amino acids).
○ The long strand of the amino acid sequence is
called primary structure.
○ If there are any changes (even minor changes) in
the primary structure it can alter the activity of the
protein.
■ Example: Hemoglobin S in sickle anemia -
(well-known example of alteration of the
primary structure)
- Replacement of a soluble glutamine
residue with a hydrophobic valine at
the sixth residue changes the nature of
the protein so that it packs aberrantly in
corpuscles and drastically alters cell
shape. Minor changes in primary
structure can have such drastic effects
because the amino acids must often
cooperate with one another to bring
about protein structure and function.
- Amino acids must open with one another
to bring out protein structure and
function.
SECONDARY STRUCTURE
• Interactions between amino acid side chains fold a
protein into predictable configurations.
○ These include ordered beta or beta-pleated sheets
and less-ordered alpha helices, or random coils
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS
○ Do not forget that the secondary structure of your
protein forms like a ribbon-like structure
composed of amino acids that are hydrogen
bonded through their side chain.
○ Hydrogen bond is formed in between the R side
chain.
● The regular folding of regions of the polypeptide
chain
TERTIARY STRUCTURE
● The 3D arrangement of all the amino acids in the
polypeptide chain
o Once there is a further folding of the amino acid
secondary structure, it will arrange to a tertiary
structure.
o Tertiary structure is the most important in the protein
function
o When the protein disappears in tertiary structure, it is
called denatured.
o Mutations in DNA that substitute different amino acids
in the primary structure can also alter tertiary
structure.
o Denatured or improperly folded proteins are not
functional.
o Proteins can also be denatured by heat (e.g., the
albumin in egg white) or by conformations forced on
innocuous peptides by infectious prions.
QUATERNARY STRUCTURE
● This is formed by the interaction of different
polypeptide chains.
● Two proteins bound together to function form a
dimer, three form a trimer, and four form a tetramer.
12
STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

● Proteins that work together in this way are called o Chromosome structure is so long or a billion sequence
oligomers, each component protein being a or length
monomer.
● This is the quaternary structure of proteins. The
combinatorial nature of protein function may
account for the genetic complexity of higher
organisms without a concurrent increase in gene
number.

CHROMOSOMES
● DNA double helix that carries genes
● Seen during cell division DEFINITION OF TERMS
● Creates identical copy of itself
● Centromere KARYOTYPE
● Individual’s collection of chromosomes
● Used to check for abnormalities
○ Laboratory technique for imaging your
chromosomes
○ There are 23 pair of chromosomes: 1 pair for sex
chromosome and 22 chromosomes for autosomal
chromosome.
o “X-like” morphology of chromosome happens during GENOTYPE
cell division ● Genetic DNA composition of organisms
o But during the phase wherein there is no cell division ○ Information about DNA
they are in linear form and seen only in cell division.
○ It is not visible and in order for it to be visible it
o Our carrying genes there are pulled off to form these
should be phenotypic.
chromosome structures
PHENOTYPE
o Our DNA in the nucleus exist as a chromatin but
reorganized as chromosome during cell division. ● Physical appearance

CHROMOSOME FORMATION
o First, the DNA begins to coil up and will wrap around
with the protein called histones.
o So, once they wrap around the protein, they will form
a bead on a string appearance which is called
nucleosome.
o As your nucleosome coils up and twists together even
more, they will produce chromatin fiber.
o And chromatin fiber will coil until they begin to have
really tight loops and they will coil up even more and
will eventually form this chromosome structure.

13
MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: NADULPIT, S.N.M.