This lecture covers current trends in molecular biology and biotechnology. It discusses the definition of molecular biology and its applications. The key components of eukaryotic cells are described, including the nucleus, organelles, plasma membrane, and genetic material DNA. Viruses, bacteria, and protozoa are also discussed. The central dogma of DNA to RNA to protein is explained. The lecture covers genetic components like genes and DNA, as well as molecular techniques like PCR and LAMP that are used to study and manipulate DNA, RNA, and proteins. The overall objectives are for participants to understand molecular biology applications and techniques and components of cells at the molecular level.

1. Lecture Title Current Trends in Molecular Biology and
Biotechnology
Lecturer Rubigilda Paraguison-Alili
Lecture Objectives  The participants are able to have a
glimpse through the current molecular
techniques and biotechnology
TOPICS
1. Biotechnology Revolution
2. Definition of Molecular Biology
3. Applications of Molecular Biology
4. The Animal and Plant Cells
5. The Virus, Bacteria and Protozoa
6. Components involved in Molecular Biology
7. Gene
8. The central Dogma in living cells
9. The Coding and Non-coding DNA
10. DNA Location
11. Basic Molecular Techniques: PCR, LAMP
12. DNA, RNA, Protein Blotting and Probing
13. Gene Expression/Cloning
14. DNA Microarray
15. RNA interference (RNAi)
16. Human Artificial Chromosome
17. Stem Cell Technology: Embryonic and Adult

2. Molecular Biology of the Cell, the Nucleic Acids
Introduction
The genetic material is defined as the substance that determines the properties or
characteristics (phenotype) of living organisms. It is also the substance that is responsible for
transferring traits and characteristics from parent to offspring. Except in some viruses where
RNA is the genetic material, the genetic material is the DNA or deoxyribonucleic acid. The
discoveries in DNA inspired more researches that led to what we now called THE CENTRAL
DOGMA, a statement of how process involving the DNA gave rise to the synthesis of the
protein. Moreover, this genetic material is also important not only in genetics but in other
applications such as in diagnostics, forensics, gene therapy and others.
Objectives: At the end of the discussion, the participants should be able to have ideas on the
current trends of Molecular Biology and Biotechnology, the Molecular Biology, its applications,
describe the major components of the cell, know fully what are nucleic acids (DNA and RNA), its
properties that is consistent with its role as the genetic material, and the principles of DNA
isolation and purification.
1. Title: Current Trends in Molecular Biology and Biotechnology
2. Biotechnology Revolution: Biological technology or Biotech is based on biology or
molecular biology particularly when used in agriculture, food science and medicine. The
United Nations Convention on Biological Diversity defined biotechnology as any
technological application that uses biological systems, living organisms, or derivatives, to
make or modify products or processes for specific use." One of the greatest global
challenges of the 21st century will be to feed, water and clothe nearly 10 billion people in
an environmentally responsible fashion. Recently, there are lots of advances in Science
and biotechnology because of the modern tool of gene technology. And biotechnology
may provide solutions to some of the challenges of the new millennium.
3. Definition of Molecular Biology
Molecular Biology is a branch of Biology that deals with the molecular basis of biological
activity. Chiefly concerns itself with understanding and the interactions between the
various systems of a cell, including the interactions between the different types of DNA,
RNA and protein biosynthesis as well as learning how these interactions are
regulated.
4. Applications of Molecular Biology
 Research
 Diagnosis
 Paternity testing
 Pedigree verification
 Forensic analysis
 Gene therapy
 Drug Design
 Genotyping

3. 5. The eukaryotic cell and its components
The cell is the basic structural and functional unit of all known living organisms. It is the
smallest unit of life that is classified as a living thing, and is often called the building
block of life
All eukaryotic cells have organelles, a nucleus, and many internal membranes. These
components divide the eukaryotic cell into sections, with each specializing in different
functions. Each function is vital to the cell's life.
 The plasma membrane serves as the selective boundary of the cell.
 The nucleus stores and protects the DNA of the cell.
 The endomembrane system consists of the endoplasmic reticulum, the Golgi
apparatus, and vesicles.
 Mitochondria transfer energy from food molecules to ATP.
6. The cell is the basic structural and functional unit of all known living organisms, in plants
and animals. It is the smallest unit of life that is classified as a living thing, and is often
called the building block of life.
 Virus- Viruses are DNA encased in protein. They are not alive, yet are capable of
replication.
7. PED Virus - The causative agent of PED is porcine epidemic diarrhea virus (PEDV) an
enveloped and single-stranded RNA virus that belongs to the family Coronaviridae. A
Corona virus that infects the cell lining of the small intestine of pigs, causing porcine
epidemic diarrhoea
8. Bacteria, Protozoa
Bacteria
 Bacteria are single celled prokaryotes.
 No organelles - only a cell wall, cell membrane, DNA, and enzymes
 Cell wall - not rigid like a plant cells, but flexible and gooey composed of peptidoglycan
 Useful as a signal to other bacteria
 Can protect pathogenic bacteria from host's defenses
 Cell membrane - semipermeable, like all cell membranes
 DNA - single loop of DNA, with numerous plasmids
Protozoa (meaning "first animals") are heterotrophic, single-celled or colonial
eukaryotes. Individuals are microscopic and range in size from a few to hundreds of
micrometers, depending on the species. Most protozoa are animal-like (heterotrophic)
because their carbon and energy must be obtained by eating or
absorbing organic compounds originating from other living organisms. As eukaryotes
they have several organelles , including at least one nucleus that contains most of the
cell's deoxyribonucleic acid (DNA).
9. Components involved in Molecular Biology
As a science that studies interactions between the molecular components that carry out
the various biological processes in living cells, an important idea in molecular biology
states that information flow in organisms follows a one-way street: Genes are
transcribed into RNA, and RNA is translated into proteins.
The molecular components make up biochemical pathways that provide the cells with
energy, facilitate processing “messages” from outside the cell itself, generate new
proteins, and replicate the cellular DNA genome. For example, molecular biologists
study how proteins interact with RNA during “translation” (the biosynthesis of new
proteins), the molecular mechanism behind DNA replication, and how genes are turned
on and off, a process called “transcription.”

4. Advances and discoveries in molecular biology continue to make major contributions to
medical research and drug development.
10. Gene : Unit of heredity
The Gene is the molecular unit of heredity of a living organism. The word is used
extensively by the scientific community for stretches of deoxyribonucleic acids (DNA)
and ribonucleic acids (RNA) that code for a polypeptide or for an RNA chain that has a
function in the organism. Living beings depend on genes, as they specify all proteins and
functional RNA chains. Genes hold the information to build and maintain an
organism's cells and pass genetic traits to offspring. All organisms have genes
corresponding to various biological traits, some of which are instantly visible, such
as eye color or number of limbs, and some of which are not, such as blood type,
increased risk for specific diseases, or the thousands of basic biochemical processes
that comprise life.
 The DNAsegments that carries genetic information are called genes.
 It is normally a stretch of DNA that codes for a type of protein or for an
RNA chain that has a function in the organism.
 Genes hold the information to build and maintain an organism's cells
and pass genetic traits to offspring.
11. The Central Dogma in Living Cells
 Genes (= DNA) the stored ‘information’
 DNA transcription is a process that involves the transcribing of genetic
information from DNA to RNA.
 In translation, mRNA along with transfer RNA (tRNA) and ribosomes work
together to produce proteins (specify amino acid sequence).
 Amino acid strings ‘fold’ into functional forms to produce the phenotype
The central dogma has also been described as "DNA makes RNA and RNA makes
protein, a positive statement which was originally termed by Crick. However, this
simplification does not make it clear that the central dogma as stated by Crick does not
preclude the reverse flow of information from RNA to DNA, only ruling out the flow from
protein to RNA or DNA. Crick's use of the word dogma was unconventional, and has
been controversial.
12. The Coding/ Non Coding DNA
 The coding DNA; the coding exons; this codes for about 20,000 -25,000 genes which in
turn code for proteins that are responsible for all the cellular processes. Exon
 The non coding DNA; non coding sequences contain information that does not lead to
the synthesis of protein. Intron
13. The DNA location
DNA is located mainly in the nucleus, but can also be found in other cell structures
called mitochondria. Since the nucleus is so small, the DNA needs to be tightly
packaged into bundles known as chromosomes.
DNA is made up of parts called nucleotides. These parts are responsible for building the
rest of the cell structures that are present in organisms. Nucleotides are made up of
phosphate groups, sugar groups and a nitrogen base. A strand of DNA is formed when

5. these nucleotides link together. The sugar groups and the phosphate groups alternate
throughout the entire strand of DNA. The nitrogen base is the base of the DNA strand
and remains constant throughout the entire strand
14. Polymerase chain reaction (PCR) –basically used to copy DNA. Different types of PCR
include reverse transcription PCR (RT-PCR) for amplification of RNA and quantitative
PCR (QPCR) to measure the amount of RNA or DNA present. Analyzed by gel
electrophoresis.
15. Loop-Mediated Isothermal Amplification (LAMP) is a single tube technique for the
amplification of DNA. A low cost alternative to detect certain diseases. It may be
combined with a reverse-transcription step to allow the detection of RNA.
16. DNA, RNA, Protein Blotting and Probing
Southern Blotting - Southern blotting was named after Edward M. Southern who
developed this procedure at Edinburgh University in the 1970s. DNA molecules are
transferred from an agarose gel onto a membrane. Designed to locate a particular
sequence of DNA within a complex mixture. For example, Southern Blotting could be
used to locate a particular gene within an entire genome.
Northern Blotting - detect specific sequences of RNA by hybridization with
complementary DNA.
Western Blotting - used to identify specific amino-acid sequences in proteins.
Eastern Blotting - used to analyze protein post translational modifications (PTM) such
as lipids, phosphomoieties and glycoconjugates. It is most often used to detect
carbohydrate epitopes.
17. Gene Expression/Cloning This technique helps scientists understand the protein
function. The DNA that codes for a particular protein is cloned or copied using PCR into
an expression vector called a plasmid. The plasmid is introduced to either an animal cell
or a bacterial cell.
The ultimate aim of expression cloning is to produce large quantities of specific proteins
or to visualize the image of your gene of interest. For example, if you want to know what
a specific mutated gene looks like on cells, you will perform gene expression analysis.
Because a gene is just a code, we want to know the picture of this mutated gene. This is
taken from the experiments that I performed in Tottori University. In this technique, DNA
coding for a protein of interest is cloned into a plasmid expression vector. This plasmid
can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells
is called transformation and introducing DNA into eukaryotic cells, such as animal cells,
is called transfection. Sometimes they use fluorescence markers to visualize the protein
of interest. I used enhanced green fluorescent protein fused with my gene of interest.
18. Neuronal cell localization of HOXA1-GFP
And the result of the expression is this. These are human neuronal cells expressing wild
type and mutated variants of HOXA1 gene. My research topic was to analyze gene
expression of mutations found in HOXA1 gene.

6. 19. HOXA1 expression- This is another type of mammalian cells expressing the same gene.
How can we utilize this technique in the future livestock biotech? We can do a lot of
assays using this technique particularly in expressing genes implicated in livestock
diseases and to test drugs and to produce enzymes.
20. DNA microarray - Commonly known as gene or genome chip, DNA chip, or gene array is
a collection of microscopic DNA spots, commonly representing genes. A DNA
microarrays or DNA chip is a collection of DNA spots mounted on a solid surface such
as a microscope slide that can be used to simultaneously quantify protein expression
levels. The technique can also be used to genotype various different genomic regions. In
spotted microarrays or two-channel or two-colour microarrays, the probes are,
cDNA or small fragments of PCR products. The cDNA from two samples to be compared
(e.g. cancer cells to normal cells) are labeled with two different fluorophores or colors
and they are mixed and hybridized to a single microarray that is then scanned in a
microarray scanner to visualize fluorescence of the two fluorophores. (Green and red).
By this, we will know which genes are up-regulated and down-regulated in cells with a
disease compared to the normal cells. One way of utilizing this is that, we can
immediately identify the genes responsible for a specific disease which will enable us to
target those genes for probably genetic manipulation and therapy.
21. RNA interference (RNAi) RNA interference is the silencing of gene expression triggered
by the presence of double-stranded RNA homologous to portions of the gene.
 In natural conditions, protects the genome from viruses, gene regulation, guides
embryonic development.
 New tool for probing how genes work and potentially for treating disease (gene
therapy).
 This is a technique involve in silencing of gene expression triggered by the
presence of dsRNA homologous to portions of the gene. The gene silencing
generally results from the cleavage and degradation of a target gene’s mRNA or
blocking the translation of intact mRNA. In a normal condition, it is very rare to
have a dsRNA in a cell. Usually they are single stranded. They are either
introduced by some viruses or by a scientist. But normally, RNAi or the presence
of dsRNA can protect the genome from viruses, for regulating genes, gene
expression or guides the embryonic development by turning down specific
genes. So, why actually would we turn down genes? For researchers, RNAi is an
exciting new tool for probing how genes work and potentially it may help in
developing treatments of some diseases.
22. The RNA interference (RNAi) process. The RNAi process begins with the presence of a
long ds-RNA molecule. An enzyme, which is called a dicer, recognizes and cuts the long
dsRNA into short 21-25bp molecules called the siRNAs. SiRNA binds to several proteins
and form an assembly called the RNA-induced silencing complex or RISC. RISC
becomes activated when the ds siRNA is unzipped which requires energy provided by
ATP. RISC can recognize and then binds to the target mRNA thereby cleaving the
mRNA causing silencing of the gene.

7. 23. RNA interference (RNAi) vs. PRRS
These data suggested that RNAi-based genetic modification might be used to breed
viral-resistant livestock with stable siRNA expression with no complications of siRNA
toxicity.
24. Human Artificial Chromosome
A human artificial chromosome (HAC) is a microchromosome that can act as a
new chromosome in a population of human cells. That is, instead of 46 chromosomes,
the cell could have 47 with the 47th being very small, roughly 6-10 megabases (Mb) in
size instead of 50-250 Mb for natural chromosomes, and able to carry new genes
introduced by human researchers. Ideally, researchers could integrate different genes
that perform a variety of functions, including disease defense.
Human artificial chromosome (HAC) can act as new chromosome
Can carry new genes around 6-10 megabases in size
Has telomere, centromere, origin of replication and sequences of DNA essential for
replication and cell division.
For ex-vivo somatic manipulation
For gene therapy
25. Human Artificial Chromosome used in calves
Aside from gene therapy in humans, we can also benefit from HAC using livestock
animals. Since chromosome and centromere structures are theoretically the same
across the mammalian species, we can introduce HAC to animals to produce our
needed enzymes or proteins. Like for instance in this paper, they introduce human
immunoglobulin gene using HAC vector into fetal fibroblasts that produce newborn
calves having human immunoglobulin.
26. Stem Cell Technology
Stem cells are primary cells found in all multi-cellular organisms. They still have the
ability differentiate into a diverse range of specialized cell types. These are cells that can
turn into any type of cells. You can turn them into neurons, cardiac cells or blood cells.
Stem cells are categorized as embryonic stem cells, which are derived from
blastocysts, and the adult stem cells, which are found in adult tissues.
Stem cells are primal cells found in all multi-cellular organisms
Have the ability to differentiate into specialized type of cells
Two Types:
1. Embryonic stem cells (blastocysts)
2. Adult stem cells (adult tissues)
27. Review: Embryology
We know that after fertilization, the zygote will form a mass of many cells to form
blastula, gastrula and neurula , forming the different germ layers: the ectoderm,
mesoderm and the endoderm. Ectoderm will form components of the skin and brain.
Mesoderm will form components of the heart, bone, kidney, blood and others and
endoderm will form components of lungs, thyroid and pancreas.
28. Embryonic Stem Cell (ES cells) are stem cells derived from the inner cell mass of a
blastocyst. ES cells are pluripotent. This means they are able to differentiate into all
derivatives the ectoderm, endoderm, and mesoderm. Because of their potentially
unlimited capacity for self-renewal, ES cell therapies have been proposed for

8. regenerative medicine and tissue replacement after injury or disease. However ES cell
technology is very controversial in human because it involves destroying of embryos, so,
until now, no approved medical treatments have been derived from embryonic stem cell
research
derived from the inner cell mass of an early stage embryo known as a blastocyst.
ES cells are pluripotent (able to become all types of cells in the body).
Not been used for therapy in human.
29. Embryonic Stem Cell Callipyge gene mutation (muscle hypertrophy) One possible
way on how we can benefit from ES cell technology is, by genetic engineering
technology, for example, we would like to manipulate gene for muscle hypertrophy
(Callipyge gene) so that your herd will have bigger muscles.
30. Adult Stem Cell/Somatic Stem Cell
The primary roles of adult stem cells in a living organism are to maintain and repair
damaged tissues. While embryonic stem cell potential remains controversial, adult stem cell
treatments are already being used to successfully treat many diseases like Parkinson's
disease, juvenile diabetes, and spinal cord injuries. They are derived with no medical risk to
the donor from blood, umbillical cord blood, bone marrow, placentas, liver, epidermis, retina,
skeletal muscle, intestine, brain, dental pulp, and fat obtained from liposuction. They can
also be derived from amnionic fluid, non-living fetal tissue and can be extracted from brains
of cadavers.
Undifferentiated cells throughout the body
Replenish and regenerate damaged tissues
Ability to divide or self renew and generate all cell types
Already being used to treat many diseases (Parkinson’s disease, diabetes, spinal cord
injuries)
blood, umbillical cord blood, bone marrow, placentas, liver, epidermis, retina, skeletal
muscle, intestine, brain, dental pulp, and fat obtained from liposuction, from amnionic
fluid, non-living fetal tissue and can be extracted from brains of cadavers.
31. Adult Stem Cell to patient with chronic heart disease.
Scientists are trying to find ways to grow adult stem cells in the lab. And, it may become
possible to generate healthy heart muscle cells in the laboratory and then transplant
those cells into patients with chronic heart disease.
32. Adult Stem Cell in Mammary gland development
33. Principles of DNA isolation and purification
DNA isolation is a process of purification of DNA from sample using a combination of
physical and chemical methods. Currently it is a routine procedure in molecular biology.
Good quality DNA is a prerequisite for all experiments of DNA manipulation. All DNA
extraction protocols comprise of the basic steps of disruption of cell membrane and
nuclear membrane to release the DNA into solution followed by precipitation of DNA
while ensuring removal of the contaminating biomolecules such as the proteins,
polysaccharides, lipids, phenols and other secondary metabolites.