Abhinav Srivastava, Ph.D

Director

Indian Biosciences and Research Institute

C-50, Sector-02, NOIDA-201310, Uttar Pradesh email: director@ibri.org.in

INTRODUCTION

The hereditary material of all multi-cellular organisms is the famous double helix of deoxyribonucleic acid (DNA), which contains all of our genes.

GENOME

The entire genetic makeup of the human cell nucleus.

All our genes together are known as “GENOME”.

HUMAN GENOME PROJECT

HUMAN GENOME PROJECT

The Human Genome Project (HGP) was the international, collaborative research program whose goal was the complete mapping and understanding of all the genes of human beings.

Genes carry the information for making all of the proteins required by the body for growth and maintenance.

Made up of ~35,000-50,000 genes which code for functional proteins in the body.

GOAL OF HGP

Identify all of the genes in human DNA.

Determine the sequence of the 3 billion chemical nucleotide bases that make up human DNA.

Store this information in data bases. Develop faster, more efficient

sequencing technologies.

GOAL OF HGP :

Develop tools for data analysis. Address the ethical, legal, and social

issues (ELSI) associated with the project.

Two Different Groups Worked to Obtain the DNA Sequence of the Human Genome

The HGP is a multinational consortium established by government research agencies and funded publicly.

Celera Genomics is a private company whose former CEO, J. Craig Venter, ran an independent sequencing project.

June 6, 2000, the HGP and Celera Genomics held a joint press conference to announce that TOGETHER they had completed ~97% of the human genome.

PUBLICATION

The International Human Genome Sequencing Consortium published their results in Nature, 409 (6822): 860-921, 2001.

“Initial Sequencing and Analysis of the Human Genome”

Celera Genomics published their results in Science, Vol 291(5507): 1304-1351, 2001. “The Sequence of the Human Genome”

METHODS AND TOOLS

STRATEGY INVOLVED

This is regarded as the most ambitious project ever undertaken by man.

Strategy may be grouped into following three stages:

i) Mappingii) Sequencingiii) Functional analysis

MAPPING

The first major goal of the project is to prepare high resolution or saturated genetic and physical maps of human genome.

Molecular markers have been used to produce maps of all the human chromosome.

By August 2000, over 9,300 markers had been mapped to particular chromosome. Therefore, any new DNA sequence (i.e., an additional marker) can be easily linked with these markers.

SEQUENCING

Determination of precise order of nucleotide in DNA.

There are following techniques for the DNA sequencing: i) Chain termination method By F.Sanger & A. R. Coulsonii) Chemical degradation method By A. Maxam & W. Gilbertiii) Automated DNA sequencing

CHAIN TERMINATION METHOD Nucleotide analogs (called dideoxynucleotides or

ddNTP) are incorporated into DNA during its synthesis together with normal nucleotides (called deoxynucleotides or dNTP).

When a ddNTP is inserted, the reaction stops = chain termination.

Four different reactions are performed. Each reaction contains either ddA, ddG, ddC, or

ddT. Autoradiography enable analysis of different

fragment lengths which correspond to different termination points.

CHEMICAL DEGRADATION METHOD Double stranded DNA fragment to be sequenced is

first labeled by attaching a radioactive phosphorous group to the 5’end of each strand.

DMSO then added and the sample heated to 90°C, degraded sample is allowed for GE.

One strand is purified from gel & divided into foru sample each of which is treated with one of the CLEAVAGE REAGENT.

The first set of reagent to be added cause a chemical modification in the nucleotide for which they are specific, making the strand susceptible to cleavage at that nucleotide when an additional chemical is added.

AUTOMATED DNA SEQUENCING This is carried out in the same way as the

chain termination method with only one difference.

Here we use FLUORESCENT LABELS to label the strand instead of radioactive label.

These labels are usually attached to the ddNTP so each chain terminated molecule carries a single label at its 3’end.

Different fluorochrome can be used for four different ddNTP.

FUNCTIONAL ANALYSIS

The ultimate objective of the HGPis to decipher the function of each of the genes estimated to be present in the human genome.

OTHER COMPLETED GENOMES

MICROBIAL GENOME

Haemophilus influenzae Escherichia coli Bacillus subtilus Helicobacter pylori Streptococcus pneumoniae AND MANY MORE !

PLANT GENOME

Arabidopsis thaliana

INSECT GENOME

Drosophila melanogaster

RODENT GENOME

Mus musculus

[ELSI]

ETHICAL, LEGAL & SOCIAL ISSUES

The U.S. Department of Energy (DOE) and the National Institutes of Health (NIH) spend between 3-5% of their annual HGP budgets toward studying the ELSI associated with availability of genetic information.

This budget is the world’s largest bioethics program, and has become a worldwide model.

EXAMPLE OF ELSI

Privacy legislation Gene testing Patenting Forensics Behavioral Genetics Genetics in the Courtroom

Who should have access to this information ?

Employers Insurers Schools Courts Adoption agencies Military

How is privacy and confidentiality managed ? Affects on society’s perceptions and

expectations of the individual Who owns genes and DNA sequences ?

The person (or company) who discovered it, or the person whose body it came from?

Should genetic information be the property of humanity?

Is it ethical to charge someone for access to a database of genetic information?

Which branches of biology will benefit from this knowledge ?

APPLICATION

MEDICINE Improvements in diagnostic and therapeutic applications

Implementation of preventative measures.

Increases in gene therapy applications.

BIOTECHNOLOGY Production of useful protein products for use in

medicine, agriculture, bioremediation and pharmaceutical industries.AntibioticsProtein replacement (factor VIII, TPA,

streptokinase, insulin, interferon…)BT insecticide toxin (from Bacillus

thuringiensis)Herbicide resistance (glyphosate resistance)Bioengineered foods [e.g. Flavr Savr tomato

(antisense – polygalacturonase) to delay rotting]

“Pharm” animals

BIOINFORMATICS The newest, fastest growing specialty in

the life sciences that integrates biotechnology and computer science.

Involved in DNA sequence assembly and analysis using computer-based techniques to determine gene function, regulation and control.

Unknown gene sequences can be compared to databases of known genes to enable similarities to lead to determination of an unknown gene’s function.

PROTEOMICS

Investigates patterns and levels of gene expression in diseased cells that can be analyzed to build databases of expression profiles.

PHARMACOGENOMICS

Investigates SNPs and DNA mutations associated with disease susceptibility and drug sensitivities.

DEVELOPMENTAL BIOLOGY

Regulation of embryonic development.

Regulation of the aging process.

EVOLUTIONARY AND COMPARATIVE BIOLOGY

Because DNA mutates at a constant rate, comparisons of DNA between different organisms can provide evolutionary histories.

SUMMARY The significance of the completion of the

human genome project cannot be overstated. With the dictionary of the genome available,

the molecular mechanisms of human health and disease will be resolved.

Armed with this knowledge a transformation in medical diagnostics and therapy is underway and will continue into the next few decades.

The application of this knowledge needs to be regulated and restricted to practices deemed ethically sound.

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3. Barnhart, Benjamin J. (1989). "DOE Human Genome Program". Human

Genome Quarterly 1: 1. Retrieved 2005-02-03. DeLisi, Charles (2001). "Genomes: 15 Years Later A Perspective by Charles

DeLisi, HGP Pioneer". Human Genome News 11: 3–4. Retrieved 2005-02-03.  International Human Genome Sequencing Consortium (2001). "Initial

sequencing and analysis of the human genome." (PDF). Nature 409: 860–921.  Venter, JC, et al. (2001). "The sequence of the human genome."

(PDF). Science 291: 1304–1351. Sanger F, Air GM, Barrell BG, et al. (February 1977). "Nucleotide sequence of

bacteriophage phi X174 DNA". Nature 265 (5596): 687–95. Waterston RH, Lander ES, Sulston JE (2003). "More on the sequencing of the

human genome". Proc Natl Acad Sci U S A. 100: 3022–4. Venter D (2003). "A Part of the Human Genome Sequence". Science 299:

1183–4. Kennedy D (2002). "Not wicked, perhaps, but tacky". Science 297: 1237.

THANK YOU !