1 bi 1 “drugs and the brain” lecture 15 thursday, april 27, 2006 the human genome for today’s...
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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.
2
from Lecture 15:
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
The Bi1 intellectual journey
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.
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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
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DNA structure
Requires Swiss-PDB viewer on your computer
http://www.its.caltech.edu/~lester/Bi-1/DNA.pdb
Compute and view H-bondsRender in solid 3-D
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Two types of base pairs in DNA: C-G pairs are more stable(Watson-Crick base pairing)
A-T base pair2 hydrogen bonds
C-G base pair3 hydrogen bonds
dA
dT
dC
dG base
ribose (sugar)
phosphate
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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.”
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no stimulus; spontaneous “miniature”postsynaptic potentials
repeated stimuli to presynaptic neuron
5 mV
50 - 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 Transmission
00.10.20.30.40.50.60.70.80.9
1
1 2 3 4 5 6Amplitude of Postsynaptic Response (mV)
Fra
ctio
n o
f O
bse
rvat
ion
s
Stimulated
Spontaneous
0 1 2 3 4 5
from Lecture 9
Therefore, Davidson
began by studying the
chemistry of DNA
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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’sby Norman Davidson
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from Lecture 15:
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG 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.
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GC content is quite nonrandom
Lander et al
Expectations from random variation:
Coefficient of variation = 100/10,000 = 1%
100000,10
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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
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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
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“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 10-16
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
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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 Mbshort arm,p
long arm,q
1 mLittle Alberts 5-13© Garland
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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
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The “Bacterial Artificial Chromosome” (BAC) ~120 kb
The Goldilocks plasmid: not too small, not too large
Mel Simon
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A single BACin a fluorescencemicroscope
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> 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
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PCR amplifies DNA exponentially
DNAsynthesis
cool tobind primers
DNAsynthesis
cool tobind primers
DNAsynthesis
cool tobind 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
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DNAsynthesis
cool tobind primers
DNAsynthesis
cool tobind primers
DNAsynthesis
cool tobind primers
fragment of DNA
to be detected
heat to separate DNA
strands
heat to separate DNA
strands
heat to separate DNA
strands
PCR amplification uses 15 to 40 cycles (3 - 5 min each) in a sealed tube
DNA polymerase (enzyme)
plusdATPdGTPdCTPdTTPDNA templateprimers
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Thermostable 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 gene on the Y chromosome; but all were positive for genes on autosomes.
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.
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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
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Proceedings of the National Academy of Sciences, 2003
Ido Braslavsky, Benedict Hebert, Emil Kartalov ‘96, Stephen R. QuakeDept 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.
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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
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Restriction enzymes cut DNA to manageable lengths
Uniqueness / fragment lengths:4-base hitter: 1 in 44 = 256 6-base hitter: 1 in 46 = 40968-base hitter: 1 in 48 = 65,536
a “6-base hitter”
Part of Little Alberts Fig. 10-4© Garland
Most restriction enzymes have 2 identical subunits
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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
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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
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Exemplar Genomes fully or partially sequenced
E. coli 4.6 ~ 4,300
Yeast 12.5 ~ 6,300
Mustard weed 120 ~ 25,000
Worm 97 ~ 22,000
Drosophila 120 ~ 15,000
Mouse 2,700 ~23,000
Human 3,300 ~23,000
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome
Rice 466 ~48,000
Mosquito 278 ~15,000
Organism genome size number of genes(common) (Mb)
See also Little Alberts 1-40
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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).
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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)
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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
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