1. The Human Genome Project Dr. Jim Whitfield, Ph.D.

2. The Human Genome Project

3. REMEMBER! It is the sequence of base pairs in DNA that determines the genetic make-up of a given organism

4. Remember If two individuals differ (and we all do) then those individuals should have different DNA sequences – at least in some portions

5. Remember These two ideas 1- The DNA sequence determines the genetic information 2- And that everybody has differing DNA were the driving forces behind the HGP and its quest to map all the genes on each human chromosome

6. Getting Started With the discovery of genetic engineering techniques such as the polymerase chain reaction (PCR) (Kary Mullis, 1983) it became possible to isolate, clone and sequence specific sections of DNA strands Because of this simple, straight forward procedure the project to sequence the entire human genome began in 1990

7. Getting Started The HGP still remains the World’s largest collaborative project

8. Getting Started The HGP was funded by the US Department of Energy, The National Institutes of Health (NIH), The Wellcome Trust in the UK, as well as several European Governments and China The project was officially completed in 2003

9. The Magnitude of the Project The human genome is estimated to have 3 x 10 9 base pairs Your book states that initial estimates were that it would cost about $3 (US) about Rs 192 to sequence each base pair, putting the total about 9 billion US dollars – However, the US Department of Energy projected the cost to be 3 billion US dollars. The final cost was 2.7 billion US dollars and it was completed 2 years ahead of schedule

10. The Magnitude of the Project Consider the massive amount of data to be generated, if each base pair represented a letter and there were a thousand letters on a page and each book contained a thousand pages it would take about 3300 books to store the DNA sequence from a single person Fortunately as the HGP advanced do did advances in computer technology which allowed for high speed storage, retrieval and analysis

11. Spin-Offs The HGP spawned the field of Bioinformatics Bioinformatics is the application of computer technology to the management of biological information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied to gene-based drug discovery and development. More about that later!

12. Goals Some of the important goals of the HGP were Identify all of the estimated 20,000 – 25,000 genes in human DNA

13. Goals Some of the important goals of the HGP were Identify all of the estimated 20,000 – 25,000 genes in human DNA Determine the sequence of the estimated 3 billion base pairs that make up human DNA

14. Goals Some of the important goals of the HGP were Identify all of the estimated 20,000 – 25,000 genes in human DNA Determine the sequence of the estimated 3 billion base pairs that make up human DNA Develop a method to store this information in a widely searchable data base (bioinformatics)

15. Goals Some of the important goals of the HGP were Identify all of the estimated 20,000 – 25,000 genes in human DNA Determine the sequence of the estimated 3 billion base pairs that make up human DNA Develop a method to store this information in a widely searchable data base (bioinformatics) Develop advanced data analysis techniques

16. Goals Continued Transfer these new technologies to other fields of industry

17. Goals Continued Address the ethical, legal, and social issues (ELSI) that would arise from the project

18. ELSI The ELSI program at NHGRI, which is considered unprecedented in biomedical science in terms of scope and level of priority, provides an effective basis from which to assess the implications of genome research, and has resulted in several notable improvements to the HGP.

19. ELSI An example is the decision to sequence the DNA of several anonymous individuals, rather than a known individual, in order to protect privacy. Craig Venter the CEO of Celara (the US Private Partner) eventually announced that he was one of the 5 people sequenced

20. ELSI The ELSI program at NHGRI, which is considered unprecedented in biomedical science in terms of scope and level of priority, provides an effective basis from which to assess the implications of genome research, and has resulted in several notable improvements to the HGP. An example is the decision to sequence the DNA of several anonymous individuals, rather than a known individual, in order to protect privacy. Craig Venter the CEO of Celara (the US Private Partner) eventually announced that he was one of the 5 people sequenced Another example is the development of widely used genetic privacy guidelines and draft legislation. The ELSI program at NHGRI now serves as a model for large, publicly funded science efforts.

21. Methodology Two distinctly different methods of sequencing were used

22. Methodology The first method focused on identifying only those genes that are expressed as RNA – This is referred to as Expressed Sequence Tags (EST’s) – Remember most DNA is Junk and does not get expressed

23. Methodology Two distinctly different methods of sequencing were used The first method focused on identifying only those genes that are expressed as RNA – This is referred to as Expressed Sequence Tags (EST’s) – Remember most DNA is Junk and does not get expressed This method limits the fragment length to between 500 and 800 nucleotides

24. Methodology The identification of ESTs has proceeded rapidly, with approximately 74.2 million ESTs now available in public databases

25. Methodology ESTs have become a tool to refine the predicted transcripts for those genes, which leads to the prediction of their protein products and ultimately their function.

26. Methodology The identification of ESTs has proceeded rapidly, with approximately 74.2 million ESTs now available in public databases ESTs have become a tool to refine the predicted transcripts for those genes, which leads to the prediction of their protein products and ultimately their function. Also, the situation in which those ESTs are obtained (tissue, organ, disease state - e.g. cancer) gives information on the conditions in which the corresponding gene is acting.

27. Methodology The second was a more of a shotgun approach who goal was to sequence the entire genome including all coding and non-coding regions. The functions of each region would be determined at a later date – This is called Sequence Annotation It is also known as DNA or Genome Annotation

28. Methodology The second was a more of a shotgun approach who goal was to sequence the entire genome including all coding and non-coding regions. The functions of each region would be determined at a later date – This is called Sequence Annotation It is also known as DNA or Genome Annotation This method does not explain what any of the DNA actually does

29. Methodology Therefore, once the DNA has been sequenced it must be broken up into smaller fragments – remember DNA is a very long polymer and there are technical limitations to how much can be sequenced at once

30. Methodology These shorter sequences are then cloned and amplified in a suitable host usually yeast or bacteria and are called YAC (yeast artificial chromosomes) and BAC (bacterial arttifical chromosomes)

31. Methodology Once the fragments have been sequenced they are arranged based on overlapping regions that are aligned by specialized computer programs

32. Methodology The sequences were then “Annotated” - describing their function The last of the human chromosomes – chromosome #1 was sequenced and annotated in 2006

33. Methodology Another major challenge encountered by the HGP researchers was being able to assign physical and and genetic maps to the genome This was accomplished using a technique known as restriction fragment length polymorphism (RFLP)

34. RFLP RFLP is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules from differing locations It also involves looking at microsatellites or Short Tandem Repeats (STR’s) – This is where short (3-5) nucleotides are repeated as often as 50X – they often exhibit high degrees of mutations

35. RFLP Analysis of RFLP variation in genomes was a vital tool in genome mapping and genetic disease analysis. If researchers were trying to initially determine the chromosomal location of a particular disease gene, they would analyze the DNA of members of a family afflicted by the disease, and look for RFLP alleles that show a similar pattern of inheritance as that of the disease. Once a disease gene was localized, RFLP analysis of other families could reveal who was at risk for the disease, or who was likely to be a carrier of the mutant genes.

36. RFLP RFLP analysis was also the basis for early methods of Genetic fingerprinting, useful in the identification of samples retrieved from crime scenes, in the determination of paternity, and in the characterization of genetic diversity or breeding patterns in animal populations.

38. America is introduced to DNA analysis and RFLP

39. What did the Human Genome Project Discover? The human genome contains 3,164, 700 nucleotide bases

40. What did the Human Genome Project Discover? The human genome contains 3,164, 700 nucleotide bases The average gene contains about 3000 bases, however there is a great range up to the largest human protein dystropin which contains 2.4 million bases (0.08% of the entire genome)

41. What did the Human Genome Project Discover? The human genome contains 3,164, 700 nucleotide bases The average gene contains about 3000 bases, however there is a great range up to the largest human protein dystropin which contains 2.4 million bases (0.08% of the entire genome) Dystrophin connects the cytoskeleton on muscle cells to the underlying cellular matrix – Deficiencies can lead to muscular dystrophy and a specific mutation leads to Duchennes muscular dystrophy

42. What did the Human Genome Project Discover? The total number of genes is approximately 30,000. This is significantly lower than the initial estimates. More than 99% of all nucleotide bases are exactly the same among all peoples

43. What did the Human Genome Project Discover? The total number of genes is approximately 30,000. This is significantly lower than the initial estimates. More than 99% of all nucleotide bases are exactly the same among all peoples

44. What did the Human Genome Project Discover? The total number of genes is approximately 30,000. This is significantly lower than the initial estimates. More than 99% of all nucleotide bases are exactly the same among all peoples The functions of many genes still remains unknown (currently about 30%)

45. What did the Human Genome Project Discover? The total number of genes is approximately 30,000. This is significantly lower than the initial estimates. More than 99% of all nucleotide bases are exactly the same among all peoples The functions of many genes still remains unknown (currently about 30%) Only about 1% of the three billion letters directly codes for proteins. Of the rest, about 25% make up genes and their regulatory elements. The function of the remaining letters is still unclear. Some of it may be redundant information left over from our evolutionary past.

46. What did the Human Genome Project Discover? Repeated sequences make up a large portion of the genome

47. What did the Human Genome Project Discover? Repeated sequences make up a large portion of the genome – approaching 98% These repeating section have no direct coding functions and they may be evolutionary relics as well as functioning in the production of ribosomal and transfer RNA

48. What did the Human Genome Project Discover? Chromosome #1 has the highest number of genes and proteins and the Y chromosome has the lowest of each

49. What did the Human Genome Project Discover? Scientists discovered about 1.4 million locations that contained single nucleotide polymorphisms (SNPs) this is where there is a difference in a single nucleotide. These differences can be used to study disease associated sequences and human evolutionary history

50. The Future The challenge to researchers and scientists now is to determine how to read the contents of the sequenced genome and then to understand how the parts work together to discover the genetic basis for health and the pathology of human disease. In this respect, genome- based research will eventually enable medical science to develop highly effective diagnostic tools, to better understand the health needs of people based on their individual genetic make-ups, and to design new and highly effective treatments for disease – so called “Precision Medicine”

51. The Future Precision medicine has lead to the new field of Pharmacogenomics. Pharmacogenomics is the study of how genes affect a person’s response to particular drugs. This relatively new field combines pharmacology (the science of drugs) and genomics (the study of genes and their functions) to develop effective, safe medications and doses that will be tailored to variations in a person’s genes.

52. India has a Bright Future Consider that