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…