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

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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 organism’s 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 (R&D) – 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 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 Types of Companies

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, Stage II- $750, 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 Founder’s 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 14 C, 35 S, 32 P, 3 H, 125 I, 131 I 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 people’s 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 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, ,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 other’s patents to exploit the invention

84 Regulatory Compliance

85 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 GLP GMP ISO 9000

86 US Regulatory Oversight in Biotech AgencyProducts 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

93 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

94 Scientific Method Codefined and promoted in 17 th 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

96 The Scientific Method

97 Scientific Method Original HypothesisDevise method to test hypothesis Analyze results Results support hypothesis Results support hypothesis but suggest minor refinements Results are so unexpected that they do not support original hypothesis and require a new hypothesis Results do not support original hypothesis but fall within range that could be expected if original hypothesis is slightly modified Retest using minor refinements of process Test using slightly modified hypothesis Test new hypotheses

98 Observe Observe Ask Questions Ask Questions Formulate Hypothesis Formulate Hypothesis Derive Predictions Formulate Hypothesis Formulate Hypothesis Derive Predictions Test Hypothesis Test Hypothesis Perform Experiments Analyze Data Test Hypothesis Test Hypothesis Perform Experiments Analyze Data Evaluate outcome Evaluate outcome Hypothesis supported Hypothesis supported Curiosity satisfied Curiosity satisfied Move onto another topic Move onto another topic No New Hypothesis New Hypothesis

99 Scientific Method & Experimental Design Testable hypothesis One variable at a time Positive controls Negative controls Background determinations Data Normalization

100 Human Genome Project

101 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

102

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

104 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.

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

106 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)

107 Construction of Genetic Linkage Map

108 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.

109 2 Types of Physical Maps Low resolution Chromosomal (Cytogenetic) map cDNA map High resolution Top-Down Mapping Bottom-up Mapping

110 Top DownBottom Up

111 Genetic Map VS. Physical Map

112

113 Automated sequencers: ABI 3700 and MegaBACE 96–well plate robotic arm and syringe 96 glass capillaries load bar

114 AUTOMATED SEQUENCE GEL

115 AUTOMATED DNA SEQUENCE

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

117 Result by the numbers The human genome contains 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 25,000— much 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.

118 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.

119 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).

120 AreaGoalAchievedDate Achieved Genetic Map2- to 5-cM resolution map (600 – 1,500 markers) 1-cM resolution map (3,000 markers) September 1994 Physical Map30,000 STSs52,000 STSsOctober 1998 DNA Sequence95% 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 < $0.25 per finished base Sequence >1,400 Mb/year at <$0.09 per finished base November 2002 Human Sequence Variation 100,000 mapped human SNPs 3.7 million mapped human SNPs February 2003 Gene IdentificationFull-length human cDNAs15,000 full-length human cDNAs March 2003 Model OrganismsComplete 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

121 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-side—has become one of the most powerful strategies for identifying human genes and interpreting their function.

122 Summary of Human Genome Project Introduction:  Background and History of HGP ( ) 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

123 Resourceful information available at: DOE website – National Human Genome Research Institute – Joint Genome Institute – National Center for Biotechnology Institute –


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