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Presenter: Viktoriia Hristova
Melbourne, Brisbane, Sydney
28 May – 18 June, 2014
Biotech Boot Camp
Session 4 – Trends and techniques
This session provides an overview of the scientific tools and
techniques used in the various biotechnology laboratories
Analysis includes key techniques pivotal to medicine,
pharmaceuticals, life sciences, agriculture, and cleantech
The session applies the techniques to specific examples
while allowing a comparison of those techniques in other
industries and in non-traditional applications
Trends and techniques
Agenda
2
• Basic overview of lab procedures
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
3
• Basic overview of lab procedures
• Biotechnology
• Molecular genetics
• Immunology
• Biochemistry
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
4
• Biotechnology: a group of diverse industries with the common link
that they apply scientific knowledge to living organisms or the
products from living organisms to benefit life on Earth
• Biologics: means the products that are produced by means of
biological processes involving DNA technology
• Traditional biotechnology industries: adopts new approaches and
modern techniques to improve the quality and productivity of their
products . These industries include Molecular Genetics, Immunology,
and Biochemistry laboratories
Trends and techniques
Basic overview of lab procedures
5
• All living things are made of cells
• Organisms can be unicellular or multicellular
• There are two types of cells
• Eukaryotes
• Prokaryotes
Just as each tissue in the human body has a function, each cell type also
has a function
• Communicate
• Grow and divide
• Manufacture proteins
Trends and techniques
Back to basics - cells
6
Trends and techniques
Cells
7
• In multicellular organisms cells communicate with other cells via
signal transduction pathways, a type of chemical message
• A target cell receives its message through proteins inserted into the
cell membrane, known as receptors
• When the signalling molecule binds to its receptor, it then transduces
an inter cellular pathway, such as transcription
• These receptor proteins, the pathways and processes they control
and the production of these proteins are mechanisms central to
biotechnology
Trends and techniques
Cellular communication
8
Trends and techniques
Cellular communications
Gene Expression
Protein Production
Signalling
Molecule
Receptor
9
• As cells grow, they produce more organelles such as mitochondria to
increase the energy of the cell
• Before a cell divides it must copy its entire genome sequence to
transfer into the new cell
• This process of cell growth and division is highly regulated and
controlled, however cancer cells do not have this level of control and
grow uncontrollably
• Cells also have a well regulated cell death process called apoptosis.
Some cancer mutations occur in these genes that control cell death
Trends and techniques
Cell division
10
Trends and techniques
Cell division
11
Trends and techniques
Cell death in healthy and cancerous cells
12 http://www.cancer.gov/cancertopics/understandingcancer/targetedtherapies/htmlcourse/page3
• Cells produce proteins
• These proteins recognise hormones, antibodies, small molecules or
other proteins
• The proteins are encoded into the cells DNA
• In order to understand the function of proteins science must
understand the genome and be able to manipulate it
Trends and techniques
Cellular mechanisms and functions
13
• At the centre of all cellular mechanisms, communication and even cell
division, is DNA
• Often referred to as the blueprint for life
• DNA is the molecule used by all organisms to store and transfer
information
• DNA is the molecule upon which the entire biotechnology industry is
built
• Particularly the understanding of how cells use DNA, and how to
manipulate DNA
Trends and techniques
The role of DNA
14
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• Immunocytochemistry
• ELISA
• Nucleic Acid Purification and Molecular Weight Determinations
• Cell Separation Methods
• Protein Separation and Quantitation
• Liquid Scintillation (double label) Counting
• Gene Expression
• Oligonucleotide Synthesis
• Autoradiography (cellular and gross)
• Restriction Enzyme Mapping
Trends and techniques
Basic overview of biotechnology lab procedures
15
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• Immunocytochemistry
• ELISA
• Nucleic Acid Purification and Molecular Weight Determinations
• Cell Separation Methods
• Protein Separation and Quantitation
• Liquid Scintillation (double label) Counting
• Gene Expression
• Oligonucleotide Synthesis
• Autoradiography (cellular and gross)
• Restriction Enzyme Mapping
Trends and techniques
Basic overview of biotechnology lab procedures
16
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• method for separation and analysis of macromolecules such as
DNA, RNA and proteins, and their fragments, based on their size
and charge
• It involves applying an electric field to move the negatively
charged molecules through a matrix of agarose gel or other
substances
• It is used in clinical chemistry to separate proteins by charge
and/or size and in biochemistry and molecular biology to
separate a mixed population of DNA and RNA fragments by
length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge
Trends and techniques
Basic overview of biotechnology lab procedures
17
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• Immunocytochemistry
• ELISA
• Nucleic Acid Purification and Molecular Weight Determinations
• Cell Separation Methods
• Protein Separation and Quantitation
• Liquid Scintillation (double label) Counting
• Gene Expression
• Oligonucleotide Synthesis
• Autoradiography (cellular and gross)
• Restriction Enzyme Mapping
Trends and techniques
Basic overview of biotechnology lab procedures
18
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Immunocytochemistry
• A technique used to assess the presence of a specific
protein or antigen in cells, by use of a specific
antibody which binds to it, thereby allowing
visualisation
Trends and techniques
Basic overview of biotechnology lab procedures
19
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• Immunocytochemistry
• ELISA
• Nucleic Acid Purification and Molecular Weight Determinations
• Cell Separation Methods
• Protein Separation and Quantitation
• Liquid Scintillation (double label) Counting
• Gene Expression
• Oligonucleotide Synthesis
• Autoradiography (cellular and gross)
• Restriction Enzyme Mapping
Trends and techniques
Basic overview of biotechnology lab procedures
20
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• ELIZA
• Enzyme-Linked ImmunoSorbant Assay is used to
determine if a particular protein is present in a sample
and if so, how much
• There are two main variations on this method:
• Measuring the amount of antibody in a sample
• Or measuring how much protein is bound by an
antibody
• This technique is central to Immunology
Trends and techniques
Basic overview of biotechnology lab procedures
21
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Gel Electrophoresis (acrylamide and agarose)
• Immunocytochemistry
• ELISA
• Nucleic Acid Purification and Molecular Weight Determinations
• Cell Separation Methods
• Protein Separation and Quantitation
• Liquid Scintillation (double label) Counting
• Gene Expression
• Oligonucleotide Synthesis
• Autoradiography (cellular and gross)
• Restriction Enzyme Mapping
Trends and techniques
Basic overview of biotechnology lab procedures
22
• Recombinant DNA Separation, and Monoclonal Antibody Procedures
• Protein Separation and Quantitation
• (or quantitative proteomics) the aim of is to obtain
quantitative information about all proteins in a sample
• It yields information about differences between
samples
• For example, this approach can be used to
compare samples from healthy and diseased
patients, while a further Immunocytochemistry or
ELIZA test will provide a qualitative result
Trends and techniques
Basic overview of biotechnology lab procedures
23
Trends and techniques
Basic overview of
recombinant DNA
lab procedures
Genetic Cloning with
bacterial plasmids
U.S. Department of Energy Genome Programs
24
Genetic cloning with bacterial plasmids
• Plasmids are small circular segments of DNA that have only a small
number of genes. Plasmids replicate separately from the bacterial
chromosome
• Foreign DNA is inserted into the plasmid, which results in a
recombinant DNA molecule. The recombinant plasmid is inserted into
the bacterium and the bacterium is allowed to replicate
• The bacterial cell divides and replicates into a clone of cells all
genetically identical to each other, and in the processes replicates the
recombinant plasmid
Trends and techniques
Basic overview of biotechnology lab procedures
25
• Overcomes the problem of source availability
• Allows the manufacture of any protein in whatever
quantity it is required
• Overcomes the problem of product safety and toxicity
• Provides an alternative to direct extraction from
inappropriate or dangerous source materials
• Facilitates the generation of newly designed proteins
Trends and techniques
Basic overview of recombinant DNA lab procedures
26
• Basic overview of lab procedures
• Biotechnology
• Molecular genetics
• Immunology
• Biochemistry
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
27
Trends and techniques
DNA
(gene)
RNA
Protein Trait
(or phenotype)
Transcription
Translation
Basic overview of molecular genetics lab procedures
Molecular Genetics is the branch of genetics concerned with the
structure and activity of genetic material at the molecular level
28
• Basic overview of lab procedures
• Biotechnology
• Molecular genetics
• Immunology
• Biochemistry
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
29
The branch of biomedicine concerned with
• the structure and function of the immune system,
• innate and acquired immunity, and
• laboratory techniques involving the interaction of antigens with
antibodies
ELISA uses the basic immunology concept of an antigen binding to its
specific antibody.
Trends and techniques
Basic overview of immunology procedures
used in labs
Antigen
Antigen Binding Site
Antibody
30
Antigens can include:
• proteins
• peptides
• hormones
• antibody
Trends and techniques
Basic overview of immunology lab procedures
Antigen
Antigen Binding Site
Antibody
31
Immunoassays
Trends and techniques
Wash
Bind capture antibody Add Antigen
Add detection AntibodyWells
Basic overview of immunology lab procedures
32
Applications
• presence of antigen
• presence of antibody
− HIV test or West Nile Virus
• food allergens
− milk, peanuts, walnuts, almonds, and eggs
Trends and techniques
Basic overview of immunology lab procedures
33
Trends and techniques
Cells - Monoclonal antibodies at work
34 http://cisncancer.org/research/new_treatments/immunotherapy/how_it_works_002.html
Gardasil
• The human papillomavirus (HPV) vaccine manufactured
by Merck and approved for preventing cervical cancer, is
produced by making a recombinant HPV protein called L1
• The recombinant L1 protein molecules reassemble to
make virus-like particles which mimic real HPV and result
in the immune system making antibodies against HPV
Trends and techniques
Cells at work – monoclonal antibodies
35
• Basic overview of lab procedures
• Biotechnology
• Molecular genetics
• Immunology
• Biochemistry
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
36
Trends and techniques
Basic overview of biochemistry lab procedures
37
Biochemistry is the study of the chemical substances and vital
processes occurring in living organisms; biological chemistry;
physiological chemistry
The production of ethanol via fermentation and anaerobic conversion is a
biochemical process that occurs naturally in micro-organisms
In recent times these organisms have become regarded as biochemical
"factories" for the treatment and conversion of biological materials into
usable energy
Biochemical conversion involves use of enzymes, bacteria or other
microorganisms to break down biomass into liquid fuels, and includes
anaerobic digestion, and fermentation
Trends and techniques
Basic overview of biochemistry lab procedures
38
The unique physicochemical and functional characteristics of
carbohydrates isolated from different sources such as corn, potato, rice
and wheat are used in a variety of biomedical and pharmaceutical
applications
Carbohydrates have properties that mimic the mechanical behaviour and
enzymatic digestibility of enzymes and proteins that control biochemical
reaction in a cell
Are used as carriers for the controlled release of drugs and other
bioactive agents, and chemically modified starches with more reactive
sites are used to carry biologically active compounds which can easily be
metabolized in the human body
Trends and techniques
Basic overview of biochemistry lab procedures
39
Along with the techniques explained in previous slides, some
further lab techniques specific to Biochemistry include:
• Northern Blotting
• Southern Blotting
• Fusion Proteins
• DNA microarray
• Bioinformatics, and
• Flow cytometry
Trends and techniques
Basic overview of biochemistry lab procedures
40
• Basic overview of lab procedures
• Research objectives of lab procedures
• Biotechnology
• Molecular Genetics
• Immunology
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
41
• Biotechnology: a group of diverse industries with the common link
that they apply scientific knowledge to living organisms or the
products from living organisms to benefit life on Earth
• Protein engineering : Design of proteins/enzymes based on
structural and mechanistic knowledge, molecular evolution,
computational design
• Synthetic biology : Creation of new biosystems (Cells and
biomolecules): Systematic, hierarchical design of artificial, bio-
inspired system using robust, standardised and well-characterised
building block
Trends and techniques
Research objectives of lab procedures used in
Biotechnology
42
Metabolic pathway engineering - design of more efficient metabolic
pathways to produce high yield of target product but low by-products
Production of biofuels from natural resources - Increase in the yield
and alcohol tolerance
• Redesign of pathway for the ethanol production in yeast to use raw
materials : corn starch, cellulose, soybean, sugar cane
• Elucidation of enzyme mechanisms
• Redesign of pathway to increase the yield and to reduce by-products
• Design of critical enzymes in the pathway
• Process development (eg: fermentation process)
• Separation and concentration
Trends and techniques
Research objectives of lab procedures used in
Molecular Genetics
43
• Determine weak spots in viruses
• Easy design of antibodies with high affinity for a target
• Design tests and techniques with low immunogenicity and
cytotoxicity
• Development of new therapeutics with high efficacy and low
side effect from non-antibody protein scaffold
• Designer therapeutic proteins
Trends and techniques
Research objectives of lab procedures used in
Immunology
44
• Basic Overview of lab procedures
• Research objectives of procedures used in
• Trends and techniques in Australia and overseas
• Case Studies
Session 4 Overview
Trends and techniques
45
Targeted therapies to treat cancer
− Roche, Genentech and ImmunoGen
− The antibody-drug conjugate field uses targeted
antibodies attached to anti-cancer toxins to home in
on tumour cells
− Roche has registered the latest win with data from a
mid-stage trial that showed that T-DM1, an antibody
linked to a toxin, outperformed the standard
treatment of two separate drugs for an advanced
form of breast cancer
Trends and techniques Australia and Overseas
46
Small molecules to treat Alzheimers
− NeuroPhage
− a new molecule it's developing based on a platform it
calls a general amyloid interaction motif, or GAIM.
GAIM works by recognizing and seeking out a
characteristic common to many toxic misfolded
proteins and targeting key stages of misfolded
protein assembly to convert toxic assemblies into
nontoxic ones, blocking the spread of misfolded
proteins within cells
Trends and techniques Australia and Overseas
47
Health care / medical devices / diagnostics
• Development of therapeutics: efficacy, toxicity
• Diagnosis: early detection and prevention of diseases
• Examples
• Biofnatics¹ – biodegradable coronary stent valve
• Encapsulate¹ – metal protein attenuating compound
Agriculture: Crop production with high yield and quality
• Examples in Australia
• Hexima – disease resistant crop
• New Natural¹ – crop fungicide
Trends and techniques Australia and Overseas
1. AusIndustry R&D Tax Incentive Biotechnology Guidance product48
Bio-based process: Pollution, CO2 emission, global
warming
• Examples in Australia:
• MBD Energy – bioremediation with algae
• Licella – biofuels from plant waste
• Biofiba – biodegradable packaging materials
• Biomine¹ – bacteria for highly saline environments
Alternative energy (Bio-energy) :
• Depletion of fossil fuels
• Use of renewable sources: Corn, sugar cane, cellulose
• Example in Australia:
• Microbiogen – fuel and feed from plant biomass
• TimbaFuels¹ – wood-waste biofuels
Trends and techniques Australia and Overseas
1. AusIndustry R&D Tax Incentive Biotechnology Guidance product49
• Basic Overview of lab procedures
• Research objectives of lab procedures
• Trends and techniques in Australia and overseas
• Case studies
Session 4 Overview
Trends and techniques
50
Trends and techniques
Case Studies and Examples
R&D Tax Incentive Biotechnology Guidance example in context
Encapsulate - Zinc metal protein attenuating powder for amyloid
plaques*
• 10 Feb 2014: Summer Street Analyst Bart Classen on Prana
Biotechnology Limited (Nasdaq: PRAN)
“We are not enthusiastic about the drug's proposed mechanism, allowing zinc
and copper ions into the cell, because there is only a limited amount of ions that
can enter the cell before they would precipitate and cause toxic deposits”
• Sangamo Biosciences early stage data shows gene therapy
approach slowed Alzheimer’s related deterioration in brain
* Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 201351
Trends and techniques
Case Studies and Examples
R&D Tax Incentive Biotechnology Guidance example in context
Encapsulate*
Development of metal protein attenuating compounds as
therapies for Parkinson’s disease
• April 2013 - Sanofi announced it will not pursue clinical trials for
Alzheimers indication until better understanding of the disease’s
mechanism exists
• March 2014 - Otsuka licenses Lundbeck’s Alzheimer’s disease
drug for up to $825M
• July 2013 - Biogen Idec in-licenses mouse model for
demyelinating diseases, another approach to Alzheimer’s, from
Myelin Research Foundation
• Dec 2013 - Eisai, Lilly form dementia consortium with
Alzheimer’s Research UK, MRC Technology
* Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 201352
Trends and techniques
Case Studies and Examples
The UPS regulatory pathway
Nurix, Inc¹
• Nurix, Inc. is a leader in discovering and developing therapies that
modulate the ubiquitin proteasome system (UPS)
• The UPS is the body's 24/7 cleaning service for the cell, degrading
and cleaning up targeted proteins inside the cell, regulating cellular
processes and maintaining protein homeostasis in a way that can
have profound implications for a wide variety of diseases
• Nurix has a particular interest in oncology and is doing R&D on the
possibilities of both speeding up (enhancing) and slowing
(inhibiting) the protein degradation process using the UPS
regulatory pathway
• Nurix has developed a high-resolution molecular understanding of
the UPS
¹ Source: Fierce Biotech 201453
Trends and techniques
Case Studies and Examples
The UPS regulatory pathway
Nurix, Inc¹
• able to engineer high selectivity small molecule drug candidates to
modulate critical ligase-substrate (protein-protein) interactions in
the UPS that drive serious diseases
• therapeutic targets previously inaccessible to small molecule drug
discovery
• initial discovery work funded by VC fund Third Rock Ventures to
get a coherent idea of the product potential and timelines involved
in the drug development while recruiting some of the top
researchers in the UPS field
• VC partner is CEO
¹ Source: Fierce Biotech 201454
Trends and techniques
Case Studies and Examples
Microarray to determine cancer
predisposition
• An array of probes capable of simultaneously
detecting multiple analytes in a sample
• In this case short, repetitive DNA sequences
but can be proteins
• Each probe is printed as a spot on the
membrane as a small spot, approximately 100
– 500 µm in diameter
• These probes or microsatellites can also be
used in forensics for paternity testing
• 2 million microsatellites in human genome
using 300,000 probes
• In this case aim is to identify unique pattern of
microsatellite variation in breast cancer
http://www.ncbi.nlm.nih.gov/55

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Nw biotech fundamentals day 1 session 4 trends and techniques

  • 1. Presenter: Viktoriia Hristova Melbourne, Brisbane, Sydney 28 May – 18 June, 2014 Biotech Boot Camp Session 4 – Trends and techniques
  • 2. This session provides an overview of the scientific tools and techniques used in the various biotechnology laboratories Analysis includes key techniques pivotal to medicine, pharmaceuticals, life sciences, agriculture, and cleantech The session applies the techniques to specific examples while allowing a comparison of those techniques in other industries and in non-traditional applications Trends and techniques Agenda 2
  • 3. • Basic overview of lab procedures • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 3
  • 4. • Basic overview of lab procedures • Biotechnology • Molecular genetics • Immunology • Biochemistry • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 4
  • 5. • Biotechnology: a group of diverse industries with the common link that they apply scientific knowledge to living organisms or the products from living organisms to benefit life on Earth • Biologics: means the products that are produced by means of biological processes involving DNA technology • Traditional biotechnology industries: adopts new approaches and modern techniques to improve the quality and productivity of their products . These industries include Molecular Genetics, Immunology, and Biochemistry laboratories Trends and techniques Basic overview of lab procedures 5
  • 6. • All living things are made of cells • Organisms can be unicellular or multicellular • There are two types of cells • Eukaryotes • Prokaryotes Just as each tissue in the human body has a function, each cell type also has a function • Communicate • Grow and divide • Manufacture proteins Trends and techniques Back to basics - cells 6
  • 8. • In multicellular organisms cells communicate with other cells via signal transduction pathways, a type of chemical message • A target cell receives its message through proteins inserted into the cell membrane, known as receptors • When the signalling molecule binds to its receptor, it then transduces an inter cellular pathway, such as transcription • These receptor proteins, the pathways and processes they control and the production of these proteins are mechanisms central to biotechnology Trends and techniques Cellular communication 8
  • 9. Trends and techniques Cellular communications Gene Expression Protein Production Signalling Molecule Receptor 9
  • 10. • As cells grow, they produce more organelles such as mitochondria to increase the energy of the cell • Before a cell divides it must copy its entire genome sequence to transfer into the new cell • This process of cell growth and division is highly regulated and controlled, however cancer cells do not have this level of control and grow uncontrollably • Cells also have a well regulated cell death process called apoptosis. Some cancer mutations occur in these genes that control cell death Trends and techniques Cell division 10
  • 12. Trends and techniques Cell death in healthy and cancerous cells 12 http://www.cancer.gov/cancertopics/understandingcancer/targetedtherapies/htmlcourse/page3
  • 13. • Cells produce proteins • These proteins recognise hormones, antibodies, small molecules or other proteins • The proteins are encoded into the cells DNA • In order to understand the function of proteins science must understand the genome and be able to manipulate it Trends and techniques Cellular mechanisms and functions 13
  • 14. • At the centre of all cellular mechanisms, communication and even cell division, is DNA • Often referred to as the blueprint for life • DNA is the molecule used by all organisms to store and transfer information • DNA is the molecule upon which the entire biotechnology industry is built • Particularly the understanding of how cells use DNA, and how to manipulate DNA Trends and techniques The role of DNA 14
  • 15. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • Immunocytochemistry • ELISA • Nucleic Acid Purification and Molecular Weight Determinations • Cell Separation Methods • Protein Separation and Quantitation • Liquid Scintillation (double label) Counting • Gene Expression • Oligonucleotide Synthesis • Autoradiography (cellular and gross) • Restriction Enzyme Mapping Trends and techniques Basic overview of biotechnology lab procedures 15
  • 16. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • Immunocytochemistry • ELISA • Nucleic Acid Purification and Molecular Weight Determinations • Cell Separation Methods • Protein Separation and Quantitation • Liquid Scintillation (double label) Counting • Gene Expression • Oligonucleotide Synthesis • Autoradiography (cellular and gross) • Restriction Enzyme Mapping Trends and techniques Basic overview of biotechnology lab procedures 16
  • 17. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • method for separation and analysis of macromolecules such as DNA, RNA and proteins, and their fragments, based on their size and charge • It involves applying an electric field to move the negatively charged molecules through a matrix of agarose gel or other substances • It is used in clinical chemistry to separate proteins by charge and/or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge Trends and techniques Basic overview of biotechnology lab procedures 17
  • 18. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • Immunocytochemistry • ELISA • Nucleic Acid Purification and Molecular Weight Determinations • Cell Separation Methods • Protein Separation and Quantitation • Liquid Scintillation (double label) Counting • Gene Expression • Oligonucleotide Synthesis • Autoradiography (cellular and gross) • Restriction Enzyme Mapping Trends and techniques Basic overview of biotechnology lab procedures 18
  • 19. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Immunocytochemistry • A technique used to assess the presence of a specific protein or antigen in cells, by use of a specific antibody which binds to it, thereby allowing visualisation Trends and techniques Basic overview of biotechnology lab procedures 19
  • 20. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • Immunocytochemistry • ELISA • Nucleic Acid Purification and Molecular Weight Determinations • Cell Separation Methods • Protein Separation and Quantitation • Liquid Scintillation (double label) Counting • Gene Expression • Oligonucleotide Synthesis • Autoradiography (cellular and gross) • Restriction Enzyme Mapping Trends and techniques Basic overview of biotechnology lab procedures 20
  • 21. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • ELIZA • Enzyme-Linked ImmunoSorbant Assay is used to determine if a particular protein is present in a sample and if so, how much • There are two main variations on this method: • Measuring the amount of antibody in a sample • Or measuring how much protein is bound by an antibody • This technique is central to Immunology Trends and techniques Basic overview of biotechnology lab procedures 21
  • 22. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Gel Electrophoresis (acrylamide and agarose) • Immunocytochemistry • ELISA • Nucleic Acid Purification and Molecular Weight Determinations • Cell Separation Methods • Protein Separation and Quantitation • Liquid Scintillation (double label) Counting • Gene Expression • Oligonucleotide Synthesis • Autoradiography (cellular and gross) • Restriction Enzyme Mapping Trends and techniques Basic overview of biotechnology lab procedures 22
  • 23. • Recombinant DNA Separation, and Monoclonal Antibody Procedures • Protein Separation and Quantitation • (or quantitative proteomics) the aim of is to obtain quantitative information about all proteins in a sample • It yields information about differences between samples • For example, this approach can be used to compare samples from healthy and diseased patients, while a further Immunocytochemistry or ELIZA test will provide a qualitative result Trends and techniques Basic overview of biotechnology lab procedures 23
  • 24. Trends and techniques Basic overview of recombinant DNA lab procedures Genetic Cloning with bacterial plasmids U.S. Department of Energy Genome Programs 24
  • 25. Genetic cloning with bacterial plasmids • Plasmids are small circular segments of DNA that have only a small number of genes. Plasmids replicate separately from the bacterial chromosome • Foreign DNA is inserted into the plasmid, which results in a recombinant DNA molecule. The recombinant plasmid is inserted into the bacterium and the bacterium is allowed to replicate • The bacterial cell divides and replicates into a clone of cells all genetically identical to each other, and in the processes replicates the recombinant plasmid Trends and techniques Basic overview of biotechnology lab procedures 25
  • 26. • Overcomes the problem of source availability • Allows the manufacture of any protein in whatever quantity it is required • Overcomes the problem of product safety and toxicity • Provides an alternative to direct extraction from inappropriate or dangerous source materials • Facilitates the generation of newly designed proteins Trends and techniques Basic overview of recombinant DNA lab procedures 26
  • 27. • Basic overview of lab procedures • Biotechnology • Molecular genetics • Immunology • Biochemistry • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 27
  • 28. Trends and techniques DNA (gene) RNA Protein Trait (or phenotype) Transcription Translation Basic overview of molecular genetics lab procedures Molecular Genetics is the branch of genetics concerned with the structure and activity of genetic material at the molecular level 28
  • 29. • Basic overview of lab procedures • Biotechnology • Molecular genetics • Immunology • Biochemistry • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 29
  • 30. The branch of biomedicine concerned with • the structure and function of the immune system, • innate and acquired immunity, and • laboratory techniques involving the interaction of antigens with antibodies ELISA uses the basic immunology concept of an antigen binding to its specific antibody. Trends and techniques Basic overview of immunology procedures used in labs Antigen Antigen Binding Site Antibody 30
  • 31. Antigens can include: • proteins • peptides • hormones • antibody Trends and techniques Basic overview of immunology lab procedures Antigen Antigen Binding Site Antibody 31
  • 32. Immunoassays Trends and techniques Wash Bind capture antibody Add Antigen Add detection AntibodyWells Basic overview of immunology lab procedures 32
  • 33. Applications • presence of antigen • presence of antibody − HIV test or West Nile Virus • food allergens − milk, peanuts, walnuts, almonds, and eggs Trends and techniques Basic overview of immunology lab procedures 33
  • 34. Trends and techniques Cells - Monoclonal antibodies at work 34 http://cisncancer.org/research/new_treatments/immunotherapy/how_it_works_002.html
  • 35. Gardasil • The human papillomavirus (HPV) vaccine manufactured by Merck and approved for preventing cervical cancer, is produced by making a recombinant HPV protein called L1 • The recombinant L1 protein molecules reassemble to make virus-like particles which mimic real HPV and result in the immune system making antibodies against HPV Trends and techniques Cells at work – monoclonal antibodies 35
  • 36. • Basic overview of lab procedures • Biotechnology • Molecular genetics • Immunology • Biochemistry • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 36
  • 37. Trends and techniques Basic overview of biochemistry lab procedures 37
  • 38. Biochemistry is the study of the chemical substances and vital processes occurring in living organisms; biological chemistry; physiological chemistry The production of ethanol via fermentation and anaerobic conversion is a biochemical process that occurs naturally in micro-organisms In recent times these organisms have become regarded as biochemical "factories" for the treatment and conversion of biological materials into usable energy Biochemical conversion involves use of enzymes, bacteria or other microorganisms to break down biomass into liquid fuels, and includes anaerobic digestion, and fermentation Trends and techniques Basic overview of biochemistry lab procedures 38
  • 39. The unique physicochemical and functional characteristics of carbohydrates isolated from different sources such as corn, potato, rice and wheat are used in a variety of biomedical and pharmaceutical applications Carbohydrates have properties that mimic the mechanical behaviour and enzymatic digestibility of enzymes and proteins that control biochemical reaction in a cell Are used as carriers for the controlled release of drugs and other bioactive agents, and chemically modified starches with more reactive sites are used to carry biologically active compounds which can easily be metabolized in the human body Trends and techniques Basic overview of biochemistry lab procedures 39
  • 40. Along with the techniques explained in previous slides, some further lab techniques specific to Biochemistry include: • Northern Blotting • Southern Blotting • Fusion Proteins • DNA microarray • Bioinformatics, and • Flow cytometry Trends and techniques Basic overview of biochemistry lab procedures 40
  • 41. • Basic overview of lab procedures • Research objectives of lab procedures • Biotechnology • Molecular Genetics • Immunology • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 41
  • 42. • Biotechnology: a group of diverse industries with the common link that they apply scientific knowledge to living organisms or the products from living organisms to benefit life on Earth • Protein engineering : Design of proteins/enzymes based on structural and mechanistic knowledge, molecular evolution, computational design • Synthetic biology : Creation of new biosystems (Cells and biomolecules): Systematic, hierarchical design of artificial, bio- inspired system using robust, standardised and well-characterised building block Trends and techniques Research objectives of lab procedures used in Biotechnology 42
  • 43. Metabolic pathway engineering - design of more efficient metabolic pathways to produce high yield of target product but low by-products Production of biofuels from natural resources - Increase in the yield and alcohol tolerance • Redesign of pathway for the ethanol production in yeast to use raw materials : corn starch, cellulose, soybean, sugar cane • Elucidation of enzyme mechanisms • Redesign of pathway to increase the yield and to reduce by-products • Design of critical enzymes in the pathway • Process development (eg: fermentation process) • Separation and concentration Trends and techniques Research objectives of lab procedures used in Molecular Genetics 43
  • 44. • Determine weak spots in viruses • Easy design of antibodies with high affinity for a target • Design tests and techniques with low immunogenicity and cytotoxicity • Development of new therapeutics with high efficacy and low side effect from non-antibody protein scaffold • Designer therapeutic proteins Trends and techniques Research objectives of lab procedures used in Immunology 44
  • 45. • Basic Overview of lab procedures • Research objectives of procedures used in • Trends and techniques in Australia and overseas • Case Studies Session 4 Overview Trends and techniques 45
  • 46. Targeted therapies to treat cancer − Roche, Genentech and ImmunoGen − The antibody-drug conjugate field uses targeted antibodies attached to anti-cancer toxins to home in on tumour cells − Roche has registered the latest win with data from a mid-stage trial that showed that T-DM1, an antibody linked to a toxin, outperformed the standard treatment of two separate drugs for an advanced form of breast cancer Trends and techniques Australia and Overseas 46
  • 47. Small molecules to treat Alzheimers − NeuroPhage − a new molecule it's developing based on a platform it calls a general amyloid interaction motif, or GAIM. GAIM works by recognizing and seeking out a characteristic common to many toxic misfolded proteins and targeting key stages of misfolded protein assembly to convert toxic assemblies into nontoxic ones, blocking the spread of misfolded proteins within cells Trends and techniques Australia and Overseas 47
  • 48. Health care / medical devices / diagnostics • Development of therapeutics: efficacy, toxicity • Diagnosis: early detection and prevention of diseases • Examples • Biofnatics¹ – biodegradable coronary stent valve • Encapsulate¹ – metal protein attenuating compound Agriculture: Crop production with high yield and quality • Examples in Australia • Hexima – disease resistant crop • New Natural¹ – crop fungicide Trends and techniques Australia and Overseas 1. AusIndustry R&D Tax Incentive Biotechnology Guidance product48
  • 49. Bio-based process: Pollution, CO2 emission, global warming • Examples in Australia: • MBD Energy – bioremediation with algae • Licella – biofuels from plant waste • Biofiba – biodegradable packaging materials • Biomine¹ – bacteria for highly saline environments Alternative energy (Bio-energy) : • Depletion of fossil fuels • Use of renewable sources: Corn, sugar cane, cellulose • Example in Australia: • Microbiogen – fuel and feed from plant biomass • TimbaFuels¹ – wood-waste biofuels Trends and techniques Australia and Overseas 1. AusIndustry R&D Tax Incentive Biotechnology Guidance product49
  • 50. • Basic Overview of lab procedures • Research objectives of lab procedures • Trends and techniques in Australia and overseas • Case studies Session 4 Overview Trends and techniques 50
  • 51. Trends and techniques Case Studies and Examples R&D Tax Incentive Biotechnology Guidance example in context Encapsulate - Zinc metal protein attenuating powder for amyloid plaques* • 10 Feb 2014: Summer Street Analyst Bart Classen on Prana Biotechnology Limited (Nasdaq: PRAN) “We are not enthusiastic about the drug's proposed mechanism, allowing zinc and copper ions into the cell, because there is only a limited amount of ions that can enter the cell before they would precipitate and cause toxic deposits” • Sangamo Biosciences early stage data shows gene therapy approach slowed Alzheimer’s related deterioration in brain * Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 201351
  • 52. Trends and techniques Case Studies and Examples R&D Tax Incentive Biotechnology Guidance example in context Encapsulate* Development of metal protein attenuating compounds as therapies for Parkinson’s disease • April 2013 - Sanofi announced it will not pursue clinical trials for Alzheimers indication until better understanding of the disease’s mechanism exists • March 2014 - Otsuka licenses Lundbeck’s Alzheimer’s disease drug for up to $825M • July 2013 - Biogen Idec in-licenses mouse model for demyelinating diseases, another approach to Alzheimer’s, from Myelin Research Foundation • Dec 2013 - Eisai, Lilly form dementia consortium with Alzheimer’s Research UK, MRC Technology * Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 201352
  • 53. Trends and techniques Case Studies and Examples The UPS regulatory pathway Nurix, Inc¹ • Nurix, Inc. is a leader in discovering and developing therapies that modulate the ubiquitin proteasome system (UPS) • The UPS is the body's 24/7 cleaning service for the cell, degrading and cleaning up targeted proteins inside the cell, regulating cellular processes and maintaining protein homeostasis in a way that can have profound implications for a wide variety of diseases • Nurix has a particular interest in oncology and is doing R&D on the possibilities of both speeding up (enhancing) and slowing (inhibiting) the protein degradation process using the UPS regulatory pathway • Nurix has developed a high-resolution molecular understanding of the UPS ¹ Source: Fierce Biotech 201453
  • 54. Trends and techniques Case Studies and Examples The UPS regulatory pathway Nurix, Inc¹ • able to engineer high selectivity small molecule drug candidates to modulate critical ligase-substrate (protein-protein) interactions in the UPS that drive serious diseases • therapeutic targets previously inaccessible to small molecule drug discovery • initial discovery work funded by VC fund Third Rock Ventures to get a coherent idea of the product potential and timelines involved in the drug development while recruiting some of the top researchers in the UPS field • VC partner is CEO ¹ Source: Fierce Biotech 201454
  • 55. Trends and techniques Case Studies and Examples Microarray to determine cancer predisposition • An array of probes capable of simultaneously detecting multiple analytes in a sample • In this case short, repetitive DNA sequences but can be proteins • Each probe is printed as a spot on the membrane as a small spot, approximately 100 – 500 µm in diameter • These probes or microsatellites can also be used in forensics for paternity testing • 2 million microsatellites in human genome using 300,000 probes • In this case aim is to identify unique pattern of microsatellite variation in breast cancer http://www.ncbi.nlm.nih.gov/55