Biology 116-Biotechnology

Ralph M. Sinibaldi, Ph.D..

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

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

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

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

“New technology is neither inherently good or harmful, this is determined by how man chooses to use the technology”

What is Biotechnology?

Biology Technology

Defining Biotechnology

Biotechnology is defined as the study and manipulation of living things or their component molecules, cells, tissues, or organs.

Biotechnology Business and Business Strategy

Fact Most new Biotech

Companies Ultimately Fail

Domains of Biotechnology. The major domains of biotechnology include 1) industrial and environmental; 2) medical/pharmaceutical; 3) agricultural; and 4) diagnostic/research

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

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

Type of companies Equipment or Instruments

• Advantages– Proprietary– Can bundle with associated reagents

• Disadvantages– Significant capital investment– Lower margins on instruments

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

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

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

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

Types of Finance Debt Financing

• Loans • Credit

Equity Financing• Private stock

– Friends & family– Private investors– Angel Investors– Venture Capital funds– Corporate partners

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

What appeals to investors Technology Business Plan Management Team Multiple Products

Compensation Salary Bonus -10 to 30 % of salary

• Must achieve aggressive goals Stock options

• Founder’s• Employee

Corporate Structure Hierarchical

Manager Manager

CCost accountant

Comptroller

XCFO

Supervvisor

Manager

DDirector of Genomics

Manager

FDirector of Mol Biol

Manager

GDirector of Chemistry

BVP of Life Science

YCSO

Supervisor

Manager

VDirector of Optics

Manager Manager

Director of Sotware

Manager

Director of Materials

AVP of Engineering

ZCTO

CEO

Corporate Structure-Matrix

Type Name HereCFO

Project ManagerProject Team A

Project ManagerProject Team B

Project ManagerProject team C

Type Name HereCSO

CEO

Corporate Structure-Hybrid Hierarchical & Matrix Combined

• Departmental Organization• Multidisciplinary Project Teams

Decision Making Technology-based

• Research• Manufacturing

Resource-based Marketing-based

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

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

Marketing SWOT analysis Strengths Weaknesses Opportunities Threats

Safety

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

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

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

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

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

Emergencies Medical response Earthquake Fire Chemical spills Regional disasters

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

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

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

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

facility

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

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

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

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

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

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

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

Radiation safety Proper training Shielding Monitoring equipment

• Geiger counter• Wipe tests

Proper storage and disposal of radioactive materials

Radiation Safety Commonly used isotopes

• 14C, 35S, 32P, 3H, 125I, 131I Geiger counters

• Different probes Scintillation Counters Radiation exposure badges

Lab Etiquette & Lab Operation

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

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

Documentation

Documentation System Corporate Policy & Procedures Department Policy & Procedures Quality System Requirements Management Control Traceability, Records & Archival

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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!

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

Intellectual Property and Compliance

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

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?

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

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

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

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

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

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

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

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

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

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

Regulatory Compliance

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 GLP

GMP ISO 9000

US Regulatory Oversight in Biotech

Agency Products RegulatedUS 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

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

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

Research and DevelopmentVocabulary

• 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

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

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

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

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

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

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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are needed to see this picture.

The Scientific Method

Scientific MethodOriginal Hypothesis Devise method to

test hypothesisAnalyze 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

ObserveObserve

Ask QuestionsAsk Questions

Formulate HypothesisFormulate Hypothesis

Derive PredictionsDerive Predictions

Test HypothesisTest Hypothesis

Perform ExperimentsPerform Experiments

Analyze DataAnalyze Data

Evaluate outcomeEvaluate outcome

Hypothesis supportedHypothesis supported

Curiosity satisfiedCuriosity satisfied

Move onto another topicMove onto another topic

No

No New Hypothesis New Hypothesis

Scientific Method & Experimental Design

Testable hypothesis One variable at a time Positive controls Negative controls Background determinations Data Normalization

Human Genome Project

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

HGP (1990 – 2003 ) Participants

US DOE

NIH

UK Medical Research Council and Wellcome Trust, UK

18 countries including France, Japan, Germany and China

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.

Methodology

DNA Source

Mapping • Genetic Linkage Map• Physical Map

DNA sequencing• Clone by clone sequencing• Whole Genome Shotgun

sequencing

Assembling

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)

Construction of Genetic Linkage Map

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.

2 Types of Physical Maps

Low resolution• Chromosomal

(Cytogenetic) map• cDNA map

High resolution• Top-Down Mapping• Bottom-up Mapping

Top Down Bottom Up

Genetic Map VS. Physical Map

Automated sequencers: ABI 3700 and MegaBACE

96–well plate

robotic arm and syringe

96 glass capillaries

load bar

AUTOMATED SEQUENCE GEL

AUTOMATED DNA SEQUENCE

Methodology DNA Source

Mapping • Genetic Linkage Map• Physical Map

DNA sequencing• Clone by clone sequencing• Whole Genome Shotgun

sequencing

Assembling• GigAssembler

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 25,000—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.

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.

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).

Area Goal Achieved Date AchievedGenetic 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 < $0.25 per finished base

Sequence >1,400Mb/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 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

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.

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 genesFuture: Applications in the field of Medicine, Forensics, Environment etc., Further research in the fields of Transcriptomics, Proteomics, Structural and

Comparative Genomics

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