development of diagnostic enzyme assay to detect leuser virus
Current Trends in Molecular Biology and BioTechnology (ppt)
1. Current Trends in
Molecular Biology and
Biotechnology
Ruby Carbonell Paraguison-Alili
Molecular Biologist
Molecular Biology Laboratory
College of Veterinary Science and Medicine
Central Luzon State University
TJCBTG∞∞
2. Biotechnology Revolution
"Biotechnology means any technological application that
uses biological systems, living organisms, or derivatives
thereof, to make or modify products or processes for
specific use.“ UNCBD (United Nations Convention on
Biological Diversity )
• Agriculture, Food Science, Medicine
Global challenge of the 21st
Century
To feed, water and clothe nearly 10 Billion
people
Biotechnology may provide solutions
3. 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.
6. 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.
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
Genomic RNA
9. Components involved in
molecular biology
•DNA- Deoxyribonucleic Acid- contains the totality of the genetic
code consisting of two long chains of nucleotides
•twisted into a double helix and joined
by hydrogen bonds between the complementary bases
•RNA- Ribonucleic Acid act as a messenger carrying instructions
from DNA for controlling the synthesis of proteins, although in
some viruses RNA rather than DNA carries the genetic
information.
•Protein composed of one or more chains of amino acids in a
specific order determined by the base sequence of nucleotides
in the DNA coding
10. Gene : Unit of heredity
• The DNA segments 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.
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
14. Basic Molecular Biotechniques
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. 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.
DNA, RNA, Protein Blotting and
Probing
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.
Expression in neuronal cells
Hoxa1 gene
Tranformation in bacterial cells
Transfection in eukaryotic cells
GFP
19. 10 His
12 His
HOXA1/Green
PBX1/
Red
7 His
MergePBX1/Red
HOXA1 expression
EGFP
COS-7
USES:
•Expressing genes
implicated in livestock
diseases
•Test drugs
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.
Spotted microarray or
2-channel or 2-colour
microarray
•Identify the genes
responsible for a specific
disease
•For gene targeting and
gene therapy
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.
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).
22. RNA interference (RNAi)
RNAi begins with the presence of a
long ds-RNA molecule
Dicer (enzyme) recognizes and cuts
dsRNA to form siRNA
siRNA bind to proteins to form
RNA-induced silencing complex
(RISC)
Unzipping of dsRNA activates RISC
RISC binds to mRNA causing
gene silencing
24. Human Artificial Chromosome
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
26. Stem Cell Technology
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)
28. Embryonic Stem Cell
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)
Genetically modified
ES cells
Blastocyst injection
Tranplantation in a
surrogate mother
1
30. Adult Stem Cell/Somatic Stem Cell
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.
32. Adult Stem Cell
As lactation
progresses, there
is also a decline
in mammary
epithelial cells.
33. Yes, we can!
Think Big! Whatever you dream or believe you can do, do it
beyond just the ordinary. And when you started it, finished it
with passion, committing everything you do to God.
34. Whatever your hand finds to do, do it
with all your might, for in the grave,
where you are going, there is neither
working nor planning nor knowledge
nor wisdom.
Ecclesiastes 9:10
35. Genesis 1:28
Then God blessed them, and God
said to them, “Be fruitful and
multiply; fill the earth and subdue
it; have dominion over the fish of
the sea, over the birds of the air,
and over every living thing that
moves on the earth.”
God needs responsible men and
women… He needs you…
