current trends in molecular biology and biotechnology (ppt)
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Current Trends in Molecular Biology and Biotechnology
Ruby Carbonell Paraguison-Alili Molecular Biologist
Molecular Biology LaboratoryCollege of Veterinary Science and Medicine
Central Luzon State UniversityTJCBTG∞∞
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 CenturyTo feed, water and clothe nearly 10 Billion peopleBiotechnology may provide solutions
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.
Applications of Molecular Biology
Research Diagnosis Paternity testingPedigree verification Forensic analysis Gene therapy Drug Design Genotyping
The Animal cell
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.
PED Virus
* The causative agent of PED isporcine 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
Bacteria, protozoa
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
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.
DNA Genes
Protein Phenotype
RNATranscription Translation
The central dogma in living cells:
ReverseTranscription (in vitro)
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
Cell
DNA LOCATION
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.
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.
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
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
EGFP (Mock)
HOXA1-WT HOXA1+1His HOXA1
+2HisHOXA1- 3His
Mutated: deletion Mutated: expansionNormal
Neuronal cell localization of HOXA1-GFP
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
DNA microarrayCommonly 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.
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).
RNA interference (RNAi)
RNAi begins with the presence of a long ds-RNA moleculeDicer (enzyme) recognizes and cuts dsRNA to form siRNA
siRNA bind to proteins to form RNA-induced silencing complex (RISC)
Unzipping of dsRNA activates RISCRISC binds to mRNA causing gene silencing
RNA interference (RNAi) vs. PRRS
Human Artificial Chromosome
Human artificial chromosome (HAC) can act as new chromosomeCan carry new genes around 6-10 megabases in sizeHas telomere, centromere, origin of replication and sequences of DNA essential for replication and cell division.For ex-vivo somatic manipulationFor gene therapy
Human Artificial Chromosome used in calves
A
Stem Cell TechnologyStem cells are primal cells found in all multi-cellular organismsHave the ability to differentiate into specialized type of cells
Two Types:1. Embryonic stem cells (blastocysts)2. Adult stem cells (adult tissues)
Review: Embryology
Embryonic Stem Cellderived 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.
Embryonic Stem Cell
Callipyge gene mutation (muscle hypertrophy)
Genetically modified ES cells
Blastocyst injection
Tranplantation in a surrogate mother
1
Adult Stem Cell/Somatic Stem CellUndifferentiated cells throughout the bodyReplenish and regenerate damaged tissuesAbility to divide or self renew and generate all cell typesAlready 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.
Adult Stem Cell
Transplant ASC to patient with chronic heart disease
Adult Stem Cell
As lactation progresses, there is also a decline in mammary epithelial cells.
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.
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
Genesis 1:28Then 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…