Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 371Bi 1 “Drugs and the Brain” Lecture 15 Thursday, April 27, 2006 The Human GenomeFor today’s lecture,It’s appropriate inspect the memorials to Norman Davidson on the walls of this lecture room.2from Lecture 15:kinasephosphorylatedproteincAMPCa2+intracellularmessengerreceptortsqiG proteinenzymechannel effectorThe Bi1 intellectual journey3Where does the Human Genome Sequencing Project stand today?Essentially finished!http://www.genome.gov/110069293.2 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.4Lander et al5Importance of DNA structureA double moleculeInformation content in base-pairingChemistry of base-pairingOrganization of the genome Lectures 15, 17, 18:Relationship between sequence and function6DNA structureRequires Swiss-PDB viewer on your computerhttp://www.its.caltech.edu/~lester/Bi-1/DNA.pdbCompute and view H-bondsRender in solid 3-D7Two types of base pairs in DNA: C-G pairs are more stable(Watson-Crick base pairing)A-T base pair2 hydrogen bondsC-G base pair3 hydrogen bondsdAdTdCdGbaseribose (sugar)phosphate8Norman 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.” 26no stimulus; spontaneous “miniature”postsynaptic potentialsrepeated stimuli to presynaptic neuron5 mV50 - 1000 channels (differs among types of synapse).This is the contents of a single vesicle.Electrophysiological analysis of quantal synaptic transmission(slide 3)Analysis of Quantal Synaptic Transmission00.10.20.30.40.50.60.70.80.911 2 3 4 5 6Amplitude of Postsynaptic Response (mV)Fraction of ObservationsStimulatedSpontaneous0 1 2 3 4 5from Lecture 9Therefore, Davidson began by studying the chemistry of DNA91. 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’sby Norman Davidson10from Lecture 15:kinasephosphorylatedproteincAMPCa2+intracellularmessengerreceptortsqiG proteinenzymechannel effectorThe Bi1 intellectual journeyBeginning in 1980, Norman Davidson used his skills at molecular biology to find the genes for several of these molecules. His intellectual journey.11GC content is quite nonrandomLander et alExpectations from random variation: Coefficient of variation = 100/10,000 = 1% 100000,10 12Humans 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 dyesB. Photoshop: we have moved the chromosomes to form pairs© Garland; Little Alberts Fig 5-1213Humans 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 dyesB. We have arranged the chromosomes to form pairs.© Garland; Little Alberts Fig 5-1214“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”, 1959.15Little Alberts Fig 10-16Genes can be localized crudely byhybridizing a fluorescent nucleotide probe to chromosomes2 m6 distinct genes are probed in this imageSeuss 195916An 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 Mbshort arm,plong arm,q1 mLittle Alberts 5-13© Garland17What happens in this room? We make enough DNA to sequence.18Two 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 pairing19The “Bacterial Artificial Chromosome” (BAC) ~120 kbThe Goldilocks plasmid: not too small, not too largeMel Simon5 A single BACin a fluorescencemicroscope20> 12 nt100 - 10,000 nucleotide pairsTwo ways to amplify a DNA sequence 2. The polymerase chain reaction (PCR)DNA polymerase requires a region of double-stranded DNALittle Alberts Fig. 10-27© Garland21PCR amplifies DNA exponentiallyDNAsynthesiscool tobind primersDNAsynthesiscool tobind primersDNAsynthesiscool tobind primersfragment of DNAto be detectedheat to separate DNAstrandsheat to separate DNAstrandsheat to separate DNAstrandsLittle Alberts Fig. 10-27-2© Garland22DNAsynthesiscool tobind primersDNAsynthesiscool tobind primersDNAsynthesiscool tobind primersfragment of DNAto be detectedheat to separate DNAstrandsheat to separate DNAstrandsheat to separate DNAstrandsPCR amplification uses 15 to 40 cycles (3 - 5 min each) in a sealed tubeDNA polymerase (enzyme)plusdATPdGTPdCTPdTTPDNA templateprimers23In fact, PCR uses dozens of sealed tubes simultaneously in a heated and cooled metal block24Thermostable DNA polymerase is obtained fromThermococcus litoralis ,an archaebacteria first isolated from deep submarine vents. This organism can grow at 98o C.Confirming that PCR can detect single molecules:In experiments on individual sperm, only 50% of the sperm had signals for a