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Biology 116-Biotechnology

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Title: Biology 116-Biotechnology


1
Biology 116-Biotechnology
  • Ralph M. Sinibaldi, Ph.D.
  • .

2
Course Goals
  • Technical training for research, development or
    production positions in biotech
  • Conceptual training in molecular biology and
    biotechnology
  • Biotech Industry overview
  • Soft skill training
  • Resumes
  • Interviews
  • Project teams and teamwork

3
Learning Outcomes
  • Describe the science of biotechnology and
    identify its product and company domains
  • Give examples of careers and job responsibilities
    associated with biotechnology
  • Understand and apply safety considerations and
    lab etiquette
  • Describe how scientific methodologies are used to
    conduct experiments and develop products
  • Understand and apply rules of documentation and
    intellectual property
  • Describe what intellectual property is and why it
    is important in biotechnology
  • Understand regulatory compliance and what
    agencies are responsible for it
  • Describe the Human Genome project and be able to
    discuss its implications

4
Vocabulary
  • Insulin a protein that facilitates the uptake
    of sugar into cells from the blood
  • DNA abbreviation for deoxyribonucleic acid, a
    double-stranded helical molecule that stores
    genetic information for the production of all of
    an organisms proteins
  • Recombinant DNA (rDNA) technology cutting and
    recombining DNA molecules
  • Polymerase chain reaction (PCR) a technique
    that involves copying short pieces of DNA and
    then making millions of copies in a short time
  • Cloning method of asexual reproduction that
    produces identical organisms
  • Fermentation a process by which, in an
    oxygen-deprived environment, a cell converts
    sugar into lactic acid or ethanol to create
    energy
  • Diabetes a disorder affecting the uptake of
    sugar by cells, due to inadequate insulin
    production or ineffective use of insulin
  • Proteases proteins whose function is to break
    down other proteins
  • Antibodies proteins developed by the immune
    system that recognize specific molecules
    (antigens)
  • Pharmaceutical relating to drugs developed for
    medical use

5
Vocabulary
  • Research and development (RD) refers to the
    early stages in product development that include
    discovery of the structure and function of a
    potential product and initial small-scale
    production
  • Pure science scientific research whose main
    purpose is to enrich the scientific knowledge
    base
  • Virus a particle containing a protein coat and
    genetic materials (either DNA or RNA) that is not
    living and requires a host to replicate
  • Applied science the practice of utilizing
    scientific knowledge for practical purposes,
    including the manufacture of a product
  • NIH abbreviation for National Institutes of
    Health the federal agency that funds and
    conducts biomedical research
  • CDC abbreviation for Centers for Disease
    Control and Prevention national research center
    for developing and applying disease prevention
    and control, environmental health, and health
    promotion and education activities to improve
    public health
  • DNA fingerprinting an experimental technique
    that is commonly used to identify individuals by
    distinguishing their unique DNA code

6
New technology is neither inherently good or
harmful, this is determined by how man chooses to
use the technology
7
What is Biotechnology?
Biology
Technology
8
Defining Biotechnology
Biotechnology is defined as the study and
manipulation of living things or their component
molecules, cells, tissues, or organs.
9
Biotechnology Business and Business Strategy
10
Fact
  • Most new Biotech Companies Ultimately Fail

11
Domains of Biotechnology. The major domains of
biotechnology include 1) industrial and
environmental 2) medical/pharmaceutical 3)
agricultural and 4) diagnostic/research
12
Types of Companies
  • Product Development
  • Advantages
  • Therapeutic products with large markets
  • Patent protection
  • High gross margins
  • Disadvantages
  • High risk
  • Long development times
  • Platform Technologies
  • Advantages
  • Shorter development times
  • Lower risk
  • Disadvantages
  • Highly competitive with ever changing technology

13
Types of Companies
  • Reagent
  • Advantages
  • Short development time
  • High profit margins
  • Disadvantages
  • May not be proprietary
  • Manufacturing costs driven
  • Service
  • Advantages
  • No manufacturing
  • Can be highly profitable
  • Disadvantages
  • Can underestimate costs

14
Type of companies
  • Equipment or Instruments
  • Advantages
  • Proprietary
  • Can bundle with associated reagents
  • Disadvantages
  • Significant capital investment
  • Lower margins on instruments

15
Starting a Company
  • An Idea or a Technology
  • Projected product(s) or service(s)
  • Market Analysis
  • Business Plan
  • Funding
  • Seed round
  • Friends Family
  • Early Venture Capital Investor
  • Angel Investor(s)
  • A round
  • Venture Capital
  • Angel Investors
  • B Round
  • Venture Capital
  • Corporate Investors or Partners
  • C Round
  • Exit Strategy
  • IPO or Acquisition

16
Business Plan
  • Summary-two pages
  • Market Opportunity
  • Company background- stage type
  • Market
  • Market analysis
  • Competitors
  • Technology
  • Proof of concept
  • Similar technologies
  • Expert opinions
  • Intellectual property
  • Patent applications
  • Potential conflicts
  • Development Plan
  • Marketing Plan
  • Distribution
  • Management
  • Org chart
  • Bios of Principals
  • Appendices

17
Role of People
  • Corporate structure
  • Skill base of employees
  • Building the right team
  • Human resources system

18
Technology
  • Publications
  • Patents
  • Proof of concept for components
  • Breadboard
  • Full Working prototype

19
Types of Finance
  • Debt Financing
  • Loans
  • Credit
  • Equity Financing
  • Private stock
  • Friends family
  • Private investors
  • Angel Investors
  • Venture Capital funds
  • Corporate partners

20
Other Sources of Funding
  • Grants
  • SBIR
  • NIH, NSF, USDA, NASA, NIST
  • Stage I- 100,000.00
  • Stage II- 750,000.00 to 1 million
  • ATP- 2 million up to 32 million
  • DARPA- national defense applications
  • Corporate partnerships
  • Marketing Distribution relationship
  • Equity

21
What appeals to investors
  • Technology
  • Business Plan
  • Management Team
  • Multiple Products

22
Compensation
  • Salary
  • Bonus -10 to 30 of salary
  • Must achieve aggressive goals
  • Stock options
  • Founders
  • Employee

23
Corporate Structure Hierarchical
24
Corporate Structure-Matrix
25
Corporate Structure-Hybrid
  • Hierarchical Matrix Combined
  • Departmental Organization
  • Multidisciplinary Project Teams

26
Decision Making
  • Technology-based
  • Research
  • Manufacturing
  • Resource-based
  • Marketing-based

27
Sustainable Business
  • Reducing Chances
  • Large and Unpredictable Capital Requirements
  • Long Product Development Cycles
  • Regulatory Issues with Product
  • Rapidly Changing Market Forces
  • High Probability of Late Stage Product Failure
  • Rare Instances of Sustained Profits
  • Increasing Chances
  • Capital Requirements Kept Low
  • Well-Defined, Predictable Business Milestones
  • Clear, Market-Oriented Business Plan
  • Critical Mass to Successfully Compete
  • Experienced Management Relevant to Strategy Being
    Pursued

28
Evolution of Company
  • Production-based
  • Technology-based
  • Market-based

29
Marketing SWOT analysis
  • Strengths
  • Weaknesses
  • Opportunities
  • Threats

30
Safety
31
Safety Values
  • Safety is not just a priority but a value
  • Safety is an unwritten rule, a special norm, the
    workers should follow in all circumstances
  • It is a value that is never questioned or
    compromised

32
Safety Habits
  • Safe for you and me
  • Prevent accidents by noticing at-risk situations
    and behaviors
  • Live safely at home, at work, and everywhere you
    go
  • Teach an attitude, promoting safety

33
Personal Safety
  • Right to work in a safe workplace
  • Responsibility
  • Protect your circle of safety and know how it may
    influence others
  • Illness and Injury prevention program

34
Work Environment
  • Organize safety for everyone
  • Remove tripping hazards
  • Do not store heavy items up high where they may
    fall
  • Do not rush or run in the workplace
  • Cleanup any liquid spills immediately
  • Report any potential hazards

35
Stress can lead to accidents
  • Recognize personal burn-out
  • Get enough sleep
  • Get professional help
  • Respect emotions of coworkers
  • Develop active listening skills
  • Develop positive, healthy relationships with
    coworkers

36
Emergencies
  • Medical response
  • Earthquake
  • Fire
  • Chemical spills
  • Regional disasters

37
What to do
  • Know emergency numbers-911 etc.
  • Be prepared and have a plan
  • Follow plan
  • Stay calm
  • Consider immediate need and response
  • Communicate with others
  • Know safety procedures, tools escape routes

38
Neighborhood or regional disaster
  • Home communication plan
  • Know alternative routes
  • Know who are your neighbors
  • Be a good citizen
  • You may have to stay where you are

39
Emergency Evacuation Plan
  • Assist those who need help to get to the
    protected area
  • Know who is present and absent
  • Communicate with other tenants
  • Be prepared for first aid and medical responses

40
Medical responses
  • Immediate first aid
  • Notify response teams, call 911
  • Provide assistance and comfort
  • Transport to trauma or urgent care facility

41
Earthquake Safety
  • Stay calm, shield yourself from falling objects
  • Prevent falling objects by storing heavy objects
    low and tie down equipment
  • Keep aisles and routes clear
  • Follow evacuation plan

42
Fire Safety
  • Report fires immediately-response time is
    critical
  • Know locations of fire fighting equipment
  • Extinguishers
  • Fire blankets
  • Fire alarm
  • Know when to evacuate get everyone out
  • If smoke is present stay low, crawl if necessary
  • Know evacuation route

43
Fire Extinguishers
  • Classification
  • A- Ordinary combustible
  • B- Flammable Liquid
  • C- Electrical
  • D- Combustible metal
  • P-A-S-S
  • Pull-Aim-Squeeze-Sweep
  • Aim at the base of the fire and sweep
  • Limited time and quantity of extinguishing
    material

44
Personal Protection
  • Actively work to prevent avoid accidents
  • Protect working space
  • Protect coworkers
  • Secondary containment- create boundaries layers
    of safety appropriate for conditions and scale of
    work

45
Working with hazards
  • Create a safety zone, CONTAIN
  • Know the hazard, PROTECT
  • Protect yourself
  • Protect those around you
  • Protect environment around you
  • Safe to touch, DECONTAMINATE
  • Secondary tertiary zones reduce the chances of
    injury or disaster

46
Personal safety attire
  • Lab coat
  • Safety glasses
  • Closed-toed shoes
  • Gloves when appropriate

47
Chemical safety
  • Know the hazards-MSDS sheets
  • Specialized training may be necessary
  • Proper storage of chemicals
  • Use proven well thought-out protocols
  • Additional personal protection attire may be
    required
  • Face shield
  • Chemical goggles
  • Latex gloves and aprons
  • Additional shielding
  • Adequate ventilation
  • Proper disposal of chemicals

48
Radiation safety
  • Proper training
  • Shielding
  • Monitoring equipment
  • Geiger counter
  • Wipe tests
  • Proper storage and disposal of radioactive
    materials

49
Radiation Safety
  • Commonly used isotopes
  • 14C, 35S, 32P, 3H, 125I, 131I
  • Geiger counters
  • Different probes
  • Scintillation Counters
  • Radiation exposure badges

50
Lab Etiquette Lab Operation
51
Common Courtesy
  • Do not use the last of a reagent and not replace
    it
  • Do not use other peoples equipment and reagents
    without asking
  • Keep your work area and common work areas clean
    and orderly
  • Do not play the radio/music without consulting
    others in the work area
  • Be willing to work as a team on all projects
  • Dress appropriately including avoiding excess
    perfume/cologne

52
Levels of Operation
  • Sterile reagents
  • Liquids autoclave at 121º C for 15-20 minutes
    using slow exhaust. Alternatively, reagents can
    be filter-sterilized using a 45 or 22 micron
    filter
  • Glassware autoclaved and cover with aluminum
    foil.
  • Plastic ware is sterile
  • Bottles/reagents may be needed to be flamed when
    opened or opened in a sterile environment
    (laminar flow hood)
  • RNase-free
  • Liquids sterilized for 1 hour or made with
    Rnase-free reagents and solvents.
  • Glassware treated in an oven for several hours
    and covered with foil
  • Reagents must be RNase-free
  • Clean room conditions
  • Dress and garb appropriately for the level of
    clean room
  • May include no makeup and cologne

53
Documentation
54
Documentation System
  • Corporate Policy Procedures
  • Department Policy Procedures
  • Quality System Requirements
  • Management Control
  • Traceability, Records Archival

55
Quality System
  • Each manufacturer shall establish and maintain a
    quality system that is appropriate for the
    specific medical device(s) designed or
    manufactured, and that meets the requirements of
    this part

56
Quality System Requirements
  • Management responsibility
  • Quality Policy- commitment to quality that is
    understood, implemented and maintained at all
    levels
  • Organization- assigned responsibility and
    independent authority, adequate resources,
    effectively establish, effectively maintain,
    review, quality plan, quality procedures
  • Quality Audit- independent documented
  • Personal- qualifications training
  • Made aware of device defects which may occur from
    improper performance of theirs specific jobs
  • Made aware of defects errors in verification
    validation

57
Quality System Subparts
  • Subpart B- Quality system requirements
  • Subpart C- Design controls
  • Subpart D- Document controls
  • Subpart E- Purchasing controls
  • Subpart F- Identification traceability
  • Subpart G- Production process controls
  • Subpart H- Acceptance activities

58
Quality System Subparts
  • Subpart I- Nonconforming product
  • Subpart J- Corrective preventive action
  • Subpart K- Labeling packaging control
  • Subpart L- Handling, storage,distribution and
    installation
  • Subpart M- Records
  • Subpart N- Servicing
  • Subpart O- Statistical techniques

59
Subpart D- Document Controls
  • Each manufacturer shall establish and maintain
    procedures to control all the documents required.
    The procedures shall provide for the following
  • Shall designate an individual(s) to review for
    adequacy and approve prior to issuance
  • Date and signatures of approval
  • Available where needed and obsolete documents
    removed
  • Changes reviewed, approve, documented, described,
    recorded, identity documents affected,communicated
    and effective date noted

60
What is a Document?
  • Legal perspective- any scrap of paper that has
    written information
  • A memo, email,letter, note, meeting minutes
  • Notebook entry, patent application, report
  • Plan, protocol, written instruction, procedure,
    policy statement
  • Label, tag, placard, sign, flowchart,
    blueprint,design description
  • Formal documentation, contracts, licenses,
    publications, marketing ads, regulatory
    submissions

61
Document Chain
  • Quality requirement, quality procedure, corporate
    policy, Mfg process, records of work, history
    files, legal contracts, Dept specific procedures,
    communication, personnel, training, reports, etc.
  • Request forms, Drafts, revision control,
    Identification, approval process,
    signatures,dates, archival, accessibility

62
Material Chain
  • Acceptable design and supply, vendor, identity,
    purchasing, receiving, inspection, acceptance,
    raw material, storage inventory, use, in-process,
    finished good, labeling, packaging,
    qualification, storage, distribution, customer,
    non-conformance, complaint,retention practices,
    disqualification, disposition, records

63
Subpart M- Records
  • All records required by this part shall be
    maintained at the manufacturing establishment or
    other location that is reasonably accessible to
    responsible officials of the manufacturer and to
    employees of FDA designated to perform
    inspections
  • Such records, including those not stored at the
    inspected establishment, shall be made readily
    available for review and copying by FDA employees
  • Such records shall be legible and shall be stored
    to minimize deterioration and to prevent loss
  • Those records stored in automated data processing
    systems shall be backed up

64
Confidentiality Retention
  • The firm should be encouraged to mark records
    they feel are confidential to assist the FDA in
    determining what information may be disclosed
    under the freedom of Information Act (FOIA)
  • Impress upon the manufacturers that marking all
    copies of records and documents confidential does
    not aid the FDA in making its FOIA determination
  • Records required by the QS/GMP must be retained
    by the manufacturer for a period of time
    equivalent to the design and expected life of the
    device, but in no case less than 2 years from the
    date of release for commercial distribution by
    the manufacturer

65
Records and Reports
  • Final report
  • Name address of facility performing study
  • Objective and procedures in approved protocol
  • Statistical methods, transformation of data,
    calculations
  • Test articles control articles (include
    stability), test system, dosage
  • Describe circumstances that may affect quality
    integrity of data
  • Name study director, other professionals,
    scientists
  • Signed and dated reports of each individual
  • Location of data records, specimens, final
    report
  • QA statement of completion
  • Signature of study director
  • Amendments to report, signed
  • Storage, retention, retrieval, of records data,
    specimens

66
Notebook Entry
  • Title, date, who, witness (legal, patent)
  • Purpose, materials methods
  • TRACEABILITY- Identify equipment, and source of
    materials protocols used
  • Factual Statements for observations and
    conclusions
  • Avoid unsupportable claims or leading suggestions
    for follow-up

67
Development Report
  • Title, project identity, investigators, date,
    distribution
  • Summarize, show linkage to records
  • Objective and outcome
  • Protocol test methods
  • The facts- results and conclusions
  • The importance (simple and realistic)

68
Validation Report
  • Title and identity, controlled document
  • Reference approved validation protocol
  • Object and outcome, clear conclusion
  • Was the method, process, product validated?
  • How?
  • Results vs acceptance parameters
  • Archive record, design history file

69
How to use documents
  • Use approved, effective documents, or documents
    identified for approved protocols
  • Follow the procedure
  • Indelible ink (black), legible, in designated
    fields for entering information
  • No extraneous entries!! Record deviations from
    procedure by creating separate document
  • Sign and date
  • The job is not finished until documented!

70
Technical Writing
  • DELIVER THE MESSAGE- communicate the objective,
    scope and outcome
  • DELIVER THE HOW- communicate the means, source of
    records, raw data and conclusions
  • DELIVER THE SO WHAT- communicate the importance
    of the findings, the relevance to the business,
    project, process or system

71
Intellectual Property and Compliance
72
Intellectual Property
  • Laboratory notebooks
  • Content Witnessing
  • Disclosures of invention
  • Priority dates
  • Confidential Information
  • Trade secret vs Patent
  • Patents
  • Compositions of matter, Process or procedure,
    Articles of Manufacture, Machines and
    Improvements
  • Types of Patents
  • Utility, Design and Plant
  • Patent Criteria
  • Conception, Reduction to practice, Utility,
    Novelty, Obviousness

73
Proper Research Notebooks
  • Physical requirements
  • Bound notebook ( no removable pages)
  • Permanent ink ( Blue or Black)
  • Content
  • Purpose of experiment
  • Materials and Methods
  • Results
  • Pictures and graphs pasted in have to be signed
    across
  • Discussion and Conclusions
  • New inventions are recorded
  • Witnessing
  • Who should witness and how often?

74
Witnessing Lab Notebooks
  • Who ?
  • Someone familiar with the research
  • It should not be a colleague working on the same
    project
  • Why not? They may be an inventor if they have
    contributed know how
  • How often?
  • Every week or two weeks

75
Disclosure of Invention
  • Some companies require as the second step in
    pursuing a patent
  • Refers to initial notebook entry
  • Can include a brief mention of related technology
    and prior art
  • Who is the inventor or inventors?
  • Inventors must contribute to the conception of
    the idea
  • People or staff who perform the experiments are
    not inventors unless they contribute
    intellectually

76
Trade Secret or Patent
  • Trade secret
  • When the process or formulation is not novel
  • When it can be easily used by competitors without
    the knowledge of inventor
  • Can last indefinitely
  • Patenting is publishing exactly how something is
    made or produced
  • Patent to protect the inventor from others using
    his invention or idea
  • Patents can be licensed to others for a fee
    and/or royalty
  • Patents are not intended to create a monopoly
  • Patents last 20 years

77
What can be patented
  • Compositions of matter
  • A new chemical entity produced from a combination
    of two or more compounds
  • Common in agricultural pharmaceutical research
  • Process or procedures
  • A series of steps that are followed to synthesize
    a new compound or make a new product
  • Articles of manufacture
  • Nearly every man-made object
  • Machines
  • Any mechanical or electrical apparatus/device
  • Improvements on any of the previous

78
Types of Patents
  • Utility Patent
  • Most common and most difficult
  • Functional characteristics of machines, devices,
    compounds
  • Exhaustive description of how to make and use the
    invention including drawings
  • Duration is 20 years
  • Design Patent
  • Protects the shape and ornamental design of an
    article
  • 14 year duration
  • Plant Patent
  • New plant variety awarded for 20 years

79
Patent Criteria
  • Conception
  • Formulation of the invention detailed enough to
    allow a person knowledgeable in the field to make
    and use the invention
  • Reduction to practice
  • Inventor makes or constructs the invention to
    demonstrate its usefullness
  • Utility
  • Invention must be useful or have utility
  • Novelty or prior art
  • Must not be a copy or a repetition of an existing
    invention
  • Obviousness
  • The invention should not be obvious to some one
    well-practiced in the field

80
Filing a Patent
  • Filing fee
  • The applicant is required to pay a fee for the
    processing of the application
  • Search examination
  • The examiner will conduct a prior art search to
    ascertain novelty and evaluate the claims to
    establish the scope of the invention
  • Publication
  • Sucessful applications will be published
  • Maintenance fees
  • Applicant must pay periodic maintenance fees

81
Parts of a Patent
  • Title
  • Inventors
  • Assignee- the company or entity who is assigned
    ownership of the patent
  • Abstract
  • Summary of invention
  • Detailed description of invention
  • Figures and drawings
  • Claims
  • Establish scope of the invention

82
Patent Strategy
  • Patenting life forms and genes
  • Easier following 1980 US Supreme court ruling,
    Diamond vs Chankrabarty
  • Reach-through patents
  • Patenting of genes based on their sequence but
    having no idea about their function
  • Patent stacking
  • Situation where more than one scientist has filed
    a patent on a gene

83
Making Money on Patents
  • Assignment- patent or patent application of
    invention can be sold or assigned to another
    party
  • License- the patent may be licensed to another
    party. This may include a licensing fee and
    royalties
  • Cross licensing- a situation where multiple
    patents cover the same or similar areas exist and
    the owners of such patents may have to cross
    license each others patents to exploit the
    invention

84
Regulatory Compliance
85
Regulatory Compliance
  • US agencies their roles
  • Food and Drug Agency (FDA)
  • GLP and GMP
  • Standard Operating Procedures (SOPs)
  • United States Dept of Agriculture (USDA-APHIS)
  • Environmental Protection Agency (EPA)
  • National Institutes of Health (NIH)
  • Office of Recombinant DNA
  • Drug Development
  • GLPGMP
  • ISO 9000

86
US Regulatory Oversight in Biotech
Agency Products Regulated
US Dept of Agriculture Plant pests, plants and veterinary biologics
Environmental Protection Agency Microbial/plant pesticides,new uses of existing pesticides, novel microorganisms
Food and Drug Administration Food, feed, food additives, veterinary drugs, human drugs, medical devices, diagnostics
87
USDA and APHIS
  • APHIS is authorized to regulate the interstate
    movement importation and field testing of
    organisms and products altered or produceds
    through biotech processes that are plant pests or
    suspected of being so.
  • Permit for movement and importation
  • Organism, origin and its intended use
  • Permit for release into environment
  • Oversight of field testing of biotech products
  • Genes and gene products, origin, purpose of test,
    experimental design,and precautions to prevent
    escape
  • Courtesy permits
  • Involves non regulated plants
  • Can involve intrastate movement

88
FDA
  • Unexpected effects- unexpected genetic effects
  • Known toxicants
  • Nutrient level
  • Allergenicity
  • New Substances
  • Antibiotic resistance selectable marker
  • Plants developed to make specialty nonfood
    substances
  • Issue specific to animal feed

89
Research and Development
Vocabulary
  • Reagent chemical used in an experiment
  • Efficacy the ability to yield a desired result
    or demonstrate that a product does what it claims
    to do
  • Large-scale production the manufacture of large
    volumes of a product
  • Clinical trials a strict series of tests that
    evaluates the effectiveness and safety of a
    medical treatment in humans
  • FDA abbreviation for the Food and Drug
    Administration the federal agency that regulates
    the use and production of food, feed, food
    additives, veterinary drugs, human drugs, and
    medical devices
  • Cystic fibrosis (CF) genetic disorder that
    clogs the respiratory and digestive systems with
    mucus
  • Therapeutic an agent that is used to treat
    diseases or disorders
  • EPA abbreviation for the Environmental
    Protection Agency the federal agency that
    enforces environmental laws including the use and
    production of microorganisms, herbicides,
    pesticides, and genetically modified
    microorganisms
  • USDA abbreviation for United States Department
    of Agriculture the federal agency that regulates
    the use and production of plants, plant products,
    plant tests, veterinary supplies and medications,
    and genetically modified plants and animals

90
Good Laboratory Practice (GLP)
  • A very consistent way of performing and
    documenting research development work
  • All documented experiments are performed in a
    consistent fashion and are witnessed in a timely
    and consistent fashion
  • Procedures are validated
  • Reagents are validated and listed
  • Instruments and equipment that are utilized in
    experiments are routinely calibrated and
    validated
  • FDA monitored

91
Good Manufacturing Practice (GMP)
  • All procedures used in manufacturing are
    consistent, fully validated and witnessed
  • Use Standard Operating Procedures (SOPs)
  • Reagents, chemicals and equipment are specified,
    validated and calibrated
  • Testing equipment specified and routinely
    calibrated
  • Some drugs need to be produced in a sterile
    environment
  • The sterility of the manufacturing environment
    needs to be monitored and documented
  • FDA monitored

92
Standard Operating Procedure
  • Detailed specific protocol
  • Steps may be monitored or witnessed
  • Reagents specified
  • Grade
  • Source or manufacturer
  • Equipment specified
  • Manufacturer
  • Model number
  • Equipment calibration
  • Calibration method
  • Calibration frequency
  • Calibration log
  • Calibrations are witnessed

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Iso 9000 or above
  • Standard ways of doing business and documenting
    it
  • In addition to manufacturing practices it can
    include
  • Shipping
  • Maintenance of plant and equipment
  • Order taking
  • Customer and technical service
  • Handling of complaints
  • Communications
  • Needed for world marketing and distribution

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Scientific Method
  • Codefined and promoted in 17th century by Rene
    Decartes and Francis Bacon
  • Steps involved in scientific method
  • Make observations
  • Ask questions
  • Make educated guesses about possible answers
  • Base predictions on the guesses
  • Devise ways to test predictions
  • Draw conclusions

95
Scientific Method
  • Hypothesis educated guess based on
    observations and questioning
  • Predicted result occurs hypothesis is most
    likely correct
  • Individuals using scientific method should be
    objective and unbiased

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The Scientific Method
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Scientific Method
Original Hypothesis
Devise method to test hypothesis
Analyze results
Results support hypothesis
Results support hypothesis but suggest minor
refinements
Results do not support original hypothesis but
fall within range that could be expected if
original hypothesis is slightly modified
Results are so unexpected that they do not
support original hypothesis and require a new
hypothesis
Retest using minor refinements of process
Test new hypotheses
Test using slightly modified hypothesis
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Observe
Ask Questions
Formulate Hypothesis Derive Predictions
Test Hypothesis Perform Experiments Analyze
Data
Evaluate outcome
No
Hypothesis supported
No
New Hypothesis
Curiosity satisfied
Move onto another topic
99
Scientific Method Experimental Design
  • Testable hypothesis
  • One variable at a time
  • Positive controls
  • Negative controls
  • Background determinations
  • Data Normalization

100
Human Genome Project
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The Human Genome Project
  • Determining the human DNA sequence
  • Understanding the function of the human genetic
    code
  • Identifying all of the genes
  • Determining their functions
  • Understanding how and when genes are turned on
    and off throughout the lifetime of an individual

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HGP (1990 2003 ) Participants
  • US DOE
  • NIH
  • UK Medical Research Council and Wellcome Trust,
    UK
  • 18 countries including France, Japan, Germany and
    China

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Goals of HGP
  1. Identify all the approximate 25,000 genes in
    human DNA.
  2. Determine the sequences of the 3 billion chemical
    base pairs that make up human DNA.
  3. Store this information in databases
  4. Improve tools for data analysis,
  5. Transfer related technologies to the private
    sector and
  6. Address the ethical, legal, and social issues
    (ELSI) that may arise from the project.

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Methodology
  • DNA Source
  • Mapping
  • Genetic Linkage Map
  • Physical Map
  • DNA sequencing
  • Clone by clone sequencing
  • Whole Genome Shotgun sequencing
  • Assembling

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Genetic Linkage Map
  • Distance between markers (genes) are determined
    by meiotic recombinational frequencies between
    the markers (or genes).
  • Gives only an estimate of the distance between
    markers or genes
  • Unit of measurement cM (centiMorgans)

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Construction of Genetic Linkage Map
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Physical Map
  • Constructed from information obtained from the
    chemical characteristics of the DNA itself and
    not from the genetic recombination analysis.
  • Unit of Measurement bp (basepair). Hence, more
    precise and exact in pinpointing the location and
    distance of the genes.

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2 Types of Physical Maps
  • Low resolution
  • Chromosomal (Cytogenetic) map
  • cDNA map
  • High resolution
  • Top-Down Mapping
  • Bottom-up Mapping

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Top Down Bottom Up
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Genetic Map VS. Physical Map
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Automated sequencers ABI 3700 and MegaBACE
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AUTOMATED SEQUENCE GEL
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AUTOMATED DNA SEQUENCE
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Methodology
  • DNA Source
  • Mapping
  • Genetic Linkage Map
  • Physical Map
  • DNA sequencing
  • Clone by clone sequencing
  • Whole Genome Shotgun sequencing
  • Assembling
  • GigAssembler

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Result by the numbers
  • The human genome contains 3164.7 million chemical
    nucleotide bases (A, C, T, and G).
  • The average gene consists of 3000 bases, but
    sizes vary greatly, with the largest known human
    gene being dystrophin at 2.4 million bases.
  • The total number of genes is estimated at 20,000
    to 25,000much lower than previous estimates of
    80,000 to 140,000.
  • Almost all (99.9) nucleotide bases are exactly
    the same in all people.
  • The functions are unknown for over 30 of
    discovered genes.

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Results Contd.
  • Less than 2 of the genome codes for proteins.
  • Repeated sequences that do not code for proteins
    ("junk DNA") make up at least 50 of the human
    genome.
  • Repetitive sequences are thought to have no
    direct functions, but they shed light on
    chromosome structure and dynamics.
  • During the past 50 million years, a dramatic
    decrease seems to have occurred in the rate of
    accumulation of repeats in the human genome.

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Genome Facts
  • The human genome's gene-dense "urban centers" are
    predominantly composed of the DNA building blocks
    G and C.
  • In contrast, the gene-poor "deserts" are rich in
    the DNA building blocks A and T.
  • Genes appear to be concentrated in random areas
    along the genome, with vast expanses of
    non-coding DNA between.
  • Stretches of up to 30,000 C and G bases repeating
    over and over often occur adjacent to gene-rich
    areas, forming a barrier between the genes and
    the "junk DNA.
  • Chromosome 1 has the most genes (2968), and the Y
    chromosome has the fewest (231).

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Area Goal Achieved Date Achieved
Genetic Map 2- to 5-cM resolution map (600 1,500 markers) 1-cM resolution map (3,000 markers) September 1994
Physical Map 30,000 STSs 52,000 STSs October 1998
DNA Sequence 95 of gene-containing part of human sequence finished to 99.99 accuracy 99 of gene-containing part of human sequence finished to 99.99 accuracy April 2003
Capacity and Cost of Finished Sequence Sequence 500 Mb/year at lt 0.25 per finished base Sequence gt1,400Mb/year at lt0.09 per finished base November 2002
Human Sequence Variation 100,000 mapped human SNPs 3.7 million mapped human SNPs February 2003
Gene Identification Full-length human cDNAs 15,000 full-length human cDNAs March 2003
Model Organisms Complete genome sequences of E. coli, S. cerevisiae, C. elegans, D. melanogaster Finished genome sequences of E. coli, S. cerevisiae, C. elegans, D. melanogaster, plus whole-genome drafts of several others, including C. briggsae, D. pseudoobscura, mouse and rat April 2003
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Endeavors after HGP
Transcriptomics - involves large-scale analysis
of messenger RNAs transcribed from active genes
to follow when, where, and under what conditions
genes are expressed. Proteomics - can bring
researchers closer to what's actually happening
in the cell than gene-expression
studies. Structural genomics - initiatives are
being launched worldwide to generate the 3-D
structures of one or more proteins from each
protein family, thus offering clues to function
and biological targets for drug design.
Comparative genomics - analyzing DNA sequence
patterns of humans and well-studied model
organisms side-by-sidehas become one of the most
powerful strategies for identifying human genes
and interpreting their function.
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Summary of Human Genome Project
  • Introduction
  • Background and History of HGP (1990-2003)
  • Methodology
  • DNA source
  • Genome Map
  • Genetic Linkage Map
  • Physical Map Low Resolution and High Resolution
  • DNA sequencing
  • Clone-by-clone sequencing
  • Whole Genome shotgun sequencing
  • Assembling
  • GigAssembler
  • Results
  • Completed Human Genome Sequencing
  • Identified 15,000 genes
  • Future
  • Applications in the field of Medicine, Forensics,
    Environment etc.,
  • Further research in the fields of
    Transcriptomics, Proteomics, Structural and
    Comparative Genomics

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Resourceful information available at
  • DOE website www.doegenomics.org
  • National Human Genome Research Institute
    www.genome.gov
  • Joint Genome Institute www.jgi.doe.gov
  • National Center for Biotechnology Institute
    www.ncbi.nlm.nih.gov
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