2. 1 Bi 1 “Drugs and the Brain” Lecture 15 Thursday, April 27, 2006 The Human Genome For today’s lecture, It’s appropriate inspect the memorials to Norman Davidson on the walls of this lecture room.

3. 2 from Lecture 15: kinase phosphorylated protein cAMP Ca 2+ intracellular messenger receptor tsqi G protein enzymechannel effector The Bi1 intellectual journey

4. 3 Where does the Human Genome Sequencing Project stand today? Essentially finished! http://www.genome.gov/11006929 3.2 billion base pairs (nearly 10 orders of magnitude) Major effort from information technology (60% of the professionals are software experts)... but annotating the human genome has just begun What is a Genome? The genome is the information set containing the totality of DNA sequence that specifies a species (on average) or an individual member of a species.

5. 4 Lander et al

6. 5 Importance of DNA structure A double molecule Information content in base-pairing Chemistry of base-pairing Organization of the genome Lectures 15, 17, 18: Relationship between sequence and function

7. 6 DNA structure Requires Swiss-PDB viewer on your computer http://www.its.caltech.edu/~lester/Bi-1/DNA.pdb Compute and view H-bonds Render in solid 3-D

8. 7 Two types of base pairs in DNA: C-G pairs are more stable (Watson-Crick base pairing) A-T base pair 2 hydrogen bonds C-G base pair 3 hydrogen bonds dA dT dC dG base ribose (sugar) phosphate

9. 8 Norman Davidson wrote, “Some time around 1958 or 1959 I was thinking about switching to biology-related research... I learned that ion channels were selective for either sodium ions or for potassium ions. This fascinated me because I knew from my undergraduate analytical chemistry course how difficult this separation was... I told Bernard Katz about my interest in doing something chemical about ion channels. He advised me to forget about it because... it would be impossible to isolate a sufficient quantity to do anything chemical.” from Lecture 9 Therefore, Davidson began by studying the chemistry of DNA

10. 9 1. The hydrogen bonds that form double-stranded DNA are easily disrupted by heating. 2. Some dyes fluoresce when they bind to double-stranded DNA. Physical Chemistry of DNA Hybridization: Studied at Caltech in ‘60’s and ‘70’s by Norman Davidson

11. 10 from Lecture 15: kinase phosphorylated protein cAMP Ca 2+ intracellular messenger receptor tsqi G protein enzymechannel effector The Bi1 intellectual journey Beginning in 1980, Norman Davidson used his skills at molecular biology to find the genes for several of these molecules. His intellectual journey.

12. 11 GC content is quite nonrandom Lander et al Expectations from random variation: Coefficient of variation = 100/10,000 = 1%

13. 12 Humans have 22 pairs of chromosomes, plus the X and Y. Males are XY; females are XX. A. Each chromosome is “painted” with a unique combination of fluorescent dyes B. Photoshop: we have moved the chromosomes to form pairs © Garland; Little Alberts Fig 5-12

14. 13 Humans have 22 pairs of chromosomes, plus the X and Y. Males are XY; females are XX. A. Each chromosome is “painted” with a unique combination of fluorescent dyes B. We have arranged the chromosomes to form pairs. © Garland; Little Alberts Fig 5-12

15. 14 “To find who’s the tallest, we start with the smallest... We start with the smallest. Then what do we do? We line them all up. Back to back. Two by two. Taller and taller. And, when we are through, we finally will find one who’s taller than who. But you have be smart and keep watching their feet. Because sometimes they stand on their tiptoes and cheat.“ Dr. Seuss explains fluorescence microscopy of chromosomes. “Happy Birthday to You”, 1959.

16. 15 Little Alberts Fig 10-16 Genes can be localized crudely by hybridizing a fluorescent nucleotide probe to chromosomes 2  m 6 distinct genes are probed in this image Seuss 1959

17. 16 An older staining method reveals dark bands in the chromosomes. (termed p12, q21, etc) The genome sequence reveals that these bands are AT-rich. #21, 45 Mb #1, 279 Mb short arm, p long arm, q 1  m Little Alberts 5-13 © Garland

18. 17 What happens in this room? We make enough DNA to sequence.

19. 18 Two ways to amplify a DNA sequence 1. Plasmid cloning in bacteria (0.500-10 kb): Little Alberts Fig. 10-22 © Garland “small, circular double-stranded DNA molecules that are separate from the larger bacterial chromosome” recombine (“splice”), with base pairing

20. 19 The “Bacterial Artificial Chromosome” (BAC) ~120 kb The Goldilocks plasmid: not too small, not too large Mel Simon 5  A single BAC in a fluorescence microscope

21. 20 > 12 nt 100 - 10,000 nucleotide pairs Two ways to amplify a DNA sequence 2. The polymerase chain reaction (PCR) DNA polymerase requires a region of double-stranded DNA Little Alberts Fig. 10-27 © Garland

22. 21 PCR amplifies DNA exponentially DNA synthesis cool to bind primers DNA synthesis cool to bind primers DNA synthesis cool to bind primers fragment of DNA to be detected heat to separate DNA strands heat to separate DNA strands heat to separate DNA strands Little Alberts Fig. 10-27-2 © Garland

23. 22 PCR amplification uses 15 to 40 cycles (3 - 5 min each) in a sealed tube DNA polymerase (enzyme) plus dATP dGTP dCTP dTTP DNA template primers

24. 23 In fact, PCR uses dozens of sealed tubes simultaneously in a heated and cooled metal block

25. 24 Thermostable DNA polymerase is obtained from Thermococcus litoralis, an archaebacteria first isolated from deep submarine vents. This organism can grow at 98 o C. Confirming that PCR can detect single molecules: In experiments on individual sperm, only 50% of the sperm had signals for a gene on the Y chromosome; but all were positive for genes on autosomes.

26. 25 Lee Hood ‘60 Fluorescence applied to DNA sequencing practical limit: 500 bp for each distinct DNA molecule The peaks broaden as nucleotides are added with statistical fluctuations around an average rate. This limits the length of each run.

27. 26 The new genome vision: “New technologies that can sequence the entire genome of any person for less than $1,000.” http://www.genome.gov/11006929

28. 27 Proceedings of the National Academy of Sciences, 2003 Ido Braslavsky, Benedict Hebert, Emil Kartalov ‘96, Stephen R. Quake Dept of Applied Physics, Caltech The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

29. 28 Total internal reflection fluorescence microscopy will enable single-molecule sequencing Dichroic mirrors Braslavsky et al, 2003 Experiments use single-molecule fluorescence, FRET, and photobleaching 10  m Condensing Lens

30. 29 Restriction enzymes cut DNA to manageable lengths Uniqueness / fragment lengths: 4-base hitter: 1 in 4 4 = 256 6-base hitter: 1 in 4 6 = 4096 8-base hitter: 1 in 4 8 = 65,536 a “6-base hitter” Part of Little Alberts Fig. 10-4 © Garland Most restriction enzymes have 2 identical subunits

31. 30 Lander et al, Figure 2 50-200 MB on each chromosome ~ 100 kB ~ 1 kB

32. 31 Cumulative pace of Disease Gene Discovery (1981-2003) Number of disease genes identified 1600 1200 800 400 0 ‘83‘85‘87‘89‘91‘93‘95‘97‘99‘01‘81‘03

33. 32 New orthologs and paralogs of common drug targets identified by searching the draft human genone sequence (Lander et al, Table 27) To be discussed in Lecture 25

34. 33 Exemplar Genomes fully or partially sequenced E. coli4.6 ~ 4,300 Yeast12.5 ~ 6,300 Mustard weed120 ~ 25,000 Worm97 ~ 22,000 Drosophila120 ~ 15,000 Mouse2,700 ~23,000 Human3,300 ~23,000 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome Rice 466 ~48,000 Mosquito 278 ~15,000 Organismgenome size number of genes (common) (Mb) See also Little Alberts 1-40

35. 34 Humans have only about as many genes as worm, and 50% more than fly. However, human genes differ in two ways from those in worm or fly. 1.Human genes are spread out over much larger regions of genomic DNA 2.Human genes are used to construct more alternative transcripts. Result: humans have ~ 5 times as many protein products as worms or flies (Lecture 17).

36. 35 More than 3 million SNPs in the human genome have been identified. This collection should allow researchers to conduct genome-wide linkage mapping of the genes in the human population. Basically: hunt for a gene for a phenotype (such as a disease) by asking which people have both the phenotype and one version of the polymorphism. This means that the phenotype is near the gene that contains the polymorphism. Single-nucleotide polymorphisms (SNPs)

37. 36 Genomics and genetics in Bi 1 “Drugs and the Brain” 15. The human genome 17.DNA to mRNA 18. From mRNA to protein 20. Genetic diversity and genetic animals 20. An Exemplar Simple Genetic Disease: Cystic Fibrosis, Cholera, and Osmosis 21.Two other exemplar simple genetic diseases: Long-QT syndrome and some Epilepsies 22. Schizophrenia and the complex genetics of psychiatric diseases 25. Evolution 1: Inferences from Molecular Biology 27. Evolution 2: The eye as an example

38. 37 Bi 1 “Drugs and the Brain” End of Lecture 15 For today’s lecture, It’s appropriate inspect the memorials to Norman Davidson on the walls of this lecture room.