Biophysics 101 Genomics and Computational Biology

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Biophysics 101 Genomics and Computational Biology
Schedule Tue Sep 19 DNA 1 Life computers
comparative genomics, databases model
utility Tue Sep 26 DNA 2 Polymorphisms,
populations, statistics, pharmacogenomics Tue Oct
03 DNA 3 Dynamic programming, Blast,
Multi-alignment, HiddenMarkovModels Tue Oct 10
RNA 1 Microarrays, library sequencing
quantitation concepts Tue Oct 17 RNA 2
Clustering by gene or condition other regulon
data sources Tue Oct 24 RNA 3 Nucleic acid
motifs the nature of biological "proofs". Tue
Oct 31 Protein 1 3D structural genomics,
homology, dynamics, function drug design Tue
Nov 07 Protein 2 Mass spectrometry,
post-synthetic modifications, Tue Nov 14
Protein 3 Quantitation of proteins, metabolites,
interactions Tue Nov 21 Network 1 Metabolic
kinetic flux balance optimization methods Tue
Nov 28 Network 2 Molecular computing,
self-assembly, genetic algorithms, neuralnets Tue
Dec 05 Network 3 Cellular, developmental,
social, ecological commercial models Tue Dec 12
Team Project presentations Tue Dec 19
Project Presentations Tue Jan 02 Project
Presentations Tue Jan 09 Project follow-up
course synthesis
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101 Section meetings
Tue 300 - 400 Haley
HMS MEC 342 Wed 700 - 800 pm
Jason HMS MEC 342 Thu
1200 - 100 Dan HMS MEC
342 (Except on 12-Oct 9-Nov he will
use MEC 338) Thu 1200 - 100
Nick HMS MEC 340 Tue 730
- 900 pm Doug Science Cntr 110 Tue
730 - 830 pm Allegra
Science Cntr 101B Tue 730 - 830 pm
Yonatan Science Cntr 102B Wed
600 - 700 pm Peter Science Cntr
112 Thu 800 - 900 pm Adnan
Science Cntr 209 Despite recruitment of new
TFs, the sections are crowded so there are no
auditor sections. Anyone registered who did not
receive email should check the list at the break.
(Email to schedule another "Biology tutorial"
for Math/CS experts)
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Last week's take home lessons
Life computers Self-assembly
Math be suspicious of approximations Catalysis
by RNA proteins "The Code" treasure (but don't
memorize) exceptions Replication
Differential equation dx/dtkx Mutation
the single molecule Noise is overcome
Human disease SNPs lt1 ppb 1.5 fold dosage
Directed graphs pedigrees Bell curve
statistics Binomial Poisson Selection
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Today's story, logic goals
Types of mutants Mutation, drift, selection
Binomial exponential dx/dt kx Association
studies c2 statistic Linked and causative
alleles Haplotypes Computing the first genome,
the second ... New technologies Random and
systematic errors
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Types of Mutants
Null PKU Dosage Trisomy 21 Conditional (e.g.
temperature or chemical) Gain of function
HbS Altered ligand specificity
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Altered specificity mutants
A consensus motif in the RFX DNA binding
domain and binding domain mutants with altered
specificity. A mutant Escherichia coli sigma
70 subunit of RNA polymerase with altered
promoter specificity. A mutant of Escherichia
coli with altered inducer specificity for the fad
regulon. A mutation in the xanthine
dehydrogenase (purine hydroxylase I) of
Aspergillus nidulans resulting in altered
specificity. Implications for the A point
mutation in the gamma2 subunit of
gamma-aminobutyric acid type A receptors results
in altered benzodiazepine binding site A point
mutation leads to altered product specificity in
beta-lactamase catalysis. A site-specific
endonuclease derived from a mutant Trp repressor
with altered DNA-binding specificity. A
spontaneous point mutation in the aac(6')-Ib'
gene results in altered substrate specificity of
aminoglycoside 6'-N-acetyltransferase of a A
streptavidin mutant with altered ligand-binding
specificity. A structural model for the HIV-1
Rev-RRE complex deduced from altered-specificity
rev variants isolated by a rapid genetic
strategy. A technique for the isolation of
yeast alcohol dehydrogenase mutants with altered
substrate specificity. A U1 small nuclear
ribonucleoprotein particle with altered
specificity induces alternative splicing of an
adenovirus E1A mRNA precursor. Amino acid
substrate specificity of Escherichia coli
phenylalanyl-tRNA synthetase altered by distinct
mutations. An altered specificity mutation in
the lambda repressor induces global
reorganization of the protein-DNA interface.
An altered-specificity mutation in a human POU
domain demonstrates functional analogy between
the POU-specific subdomain and Analysis of
estrogen response element binding by genetically
selected steroid receptor DNA binding domain
mutants exhibiting altered Antiprotease
targeting altered specificity of alpha
1-antitrypsin by amino acid replacement at the
reactive centre. AraC proteins with altered
DNA sequence specificity which activate a mutant
promoter in Escherichia coli. Assessment of
the role of an omega loop of cholesterol oxidase
a truncated loop mutant has altered substrate
specificity. Butyramide-utilizing mutants of
Pseudomonas aeruginosa 8602 which produce an
amidase with altered substrate specificity.
Carboxyl-terminal domain dimer interface mutant
434 repressors have altered dimerization and DNA
binding specificities. Characterization of the
nuclear protein import mechanism using Ran
mutants with altered nucleotide binding
specificities. Computational method for the
design of enzymes with altered substrate
specificity. Crystallographic analysis of
trypsin-G226A. A specificity pocket mutant of rat
trypsin with altered binding and catalysis.
Designing zinc-finger ADR1 mutants with altered
specificity of DNA binding to T in UAS1
sequences. Dinitrogenase with altered
substrate specificity results from the use of
homocitrate analogues for in vitro synthesis of
the iron-molybdenum Dissecting Fas signaling
with an altered-specificity death-domain mutant
requirement of FADD binding for apoptosis but not
Jun DNA-binding-defective mutants of the
Epstein-Barr virus lytic switch activator Zta
transactivate with altered specificities.
E461H-beta-galactosidase (Escherichia coli)
altered divalent metal specificity and slow but
reversible metal inactivation. EcoRV-T94V a
mutant restriction endonuclease with an altered
substrate specificity towards modified
oligodeoxynucleotides. Engineering proteases
with altered specificity. Engrailed
(Gln50--gtLys) homeodomain-DNA complex at 1.9 A
resolution structural basis for enhanced
affinity and altered specificity. Enhanced
activity and altered specificity of phospholipase
A2 by deletion of a surface loop. Escherichia
coli hemolysin mutants with altered target cell
specificity. Evidence for an altered operator
specificity catabolite repression control of the
leucine operon in Salmonella typhimurium.
Evidence that HT mutant strains of bacteriophage
P22 retain an altered form of substrate
specificity in the formation of transducing
Ferrichrome transport in Escherichia coli K-12
altered substrate specificity of mutated
periplasmic FhuD and interaction of FhuD with
the Generation of estrogen receptor mutants
with altered ligand specificity for use in
establishing a regulatable gene expression
system.
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Altered specificity mutants (continued)
Genetic strategy for analyzing specificity of
dimer formation Escherichia coli cyclic AMP
receptor protein mutant altered in dimerization
Immunoglobulin V region variants in hybridoma
cells. I. Isolation of a variant with altered
idiotypic and antigen binding specificity. In
vitro selection for altered divalent metal
specificity in the RNase P RNA. In vitro
selection of zinc fingers with altered
DNA-binding specificity. In vivo selection of
basic region-leucine zipper proteins with altered
DNA-binding specificities. Isolation and
properties of Escherichia coli ATPase mutants
with altered divalent metal specificity for ATP
hydrolysis. Isolation of altered specificity
mutants of the single-chain 434 repressor that
recognize asymmetric DNA sequences containing
TTAA Mechanisms of spontaneous mutagenesis
clues from altered mutational specificity in DNA
repair-defective strains. Molecular basis of
altered enzyme specificities in a family of
mutant amidases from Pseudomonas aeruginosa.
Mutants in position 69 of the Trp repressor of
Escherichia coli K12 with altered DNA-binding
specificity. Mutants of eukaryotic initiation
factor eIF-4E with altered mRNA cap binding
specificity reprogram mRNA selection by ribosomes
in Mutational analysis of the CitA citrate
transporter from Salmonella typhimurium altered
substrate specificity. Na-coupled transport
of melibiose in Escherichia coli analysis of
mutants with altered cation specificity.
Nuclease activities of Moloney murine leukemia
virus reverse transcriptase. Mutants with altered
substrate specificities. Probing the altered
specificity and catalytic properties of mutant
subtilisin chemically modified at position S156C
and S166C in the S1 Products of alternatively
spliced transcripts of the Wilms' tumor
suppressor gene, wt1, have altered DNA binding
specificity and regulate Proline transport in
Salmonella typhimurium putP permease mutants
with altered substrate specificity. Random
mutagenesis of the substrate-binding site of a
serine protease can generate enzymes with
increased activities and altered Redesign of
soluble fatty acid desaturases from plants for
altered substrate specificity and double bond
position. Selection and characterization of
amino acid substitutions at residues 237-240 of
TEM-1 beta-lactamase with altered substrate
specificity Selection strategy for site-directed
mutagenesis based on altered beta-lactamase
specificity. Site-directed mutagenesis of
yeast eEF1A. Viable mutants with altered
nucleotide specificity. Structure and dynamics
of the glucocorticoid receptor DNA-binding
domain comparison of wild type and a mutant with
altered specificity. Structure-function
analysis of SH3 domains SH3 binding specificity
altered by single amino acid substitutions.
Sugar-binding and crystallographic studies of an
arabinose-binding protein mutant (Met108Leu) that
exhibits enhanced affinity altered T7 RNA
polymerase mutants with altered promoter
specificities. The specificity of
carboxypeptidase Y may be altered by changing the
hydrophobicity of the S'1 binding pocket. The
structural basis for the altered substrate
specificity of the R292D active site mutant of
aspartate aminotransferase from E. coli.
Thymidine kinase with altered substrate
specificity of acyclovir resistant
varicella-zoster virus. U1 small nuclear RNAs
with altered specificity can be stably expressed
in mammalian cells and promote permanent changes
in Use of altered specificity mutants to probe
a specific protein-protein interaction in
differentiation the GATA-1FOG complex. Use
of Chinese hamster ovary cells with altered
glycosylation patterns to define the carbohydrate
specificity of Entamoeba histolytica Using
altered specificity Oct-1 and Oct-2 mutants to
analyze the regulation of immunoglobulin gene
transcription. Variants of subtilisin BPN'
with altered specificity profiles. Yeast and
human TFIID with altered DNA-binding specificity
for TATA elements.
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From genomics to public health

• Vaccines, drugs, lifestyle, public health
  measures
• Pharmacogenomics
• Targets (proteins or phenotypes)
• Chemical diversity
• Gene therapy, DNA vaccines, ribozymes, nutrition
  
• High-throughput screening of compounds
• Animal testing
• Clinical trials phase 1,2,3
• Formulation Bioavailability
• Toxicity
• Delivery time release ,feedback
• Marketing and societal priorities

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Pharmacogenomics
Gene/Enzyme Drug
Quantitative effect
Examples of clinically relevant genetic
polymorphisms influencing drug metabolism and
effects. Additional data
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Diversity Databases

• 45 genomes completed 324 started
  216.190.101.28/GOLD
• (DBCat NAR) 513 bio-databases plus
• List of SNP databasesariel.ucs.unimelb.edu.au80/
  cotton/mdi.htm
• 803557 human SNPs www.ncbi.nlm.nih.gov/SNP
  296990 mapped snp.cshl.org
• 21591 SNPs in genes http//www.uwcm.ac.uk/uwcm/mg/
  hgmd0.html

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(No Transcript)
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A significant basepair
aggtcatctgagGtcaggagttca ANALYSIS ALU
repeat found upstream of
Iodothyronine deiodinase, Myeloperoxidase,
Keratin K18, HoxA1,etc. "-463 G creates a
stronger SP1 binding site retinoic acid
response element (RARE) in the allele...
overrepresented in acute promyelocytic
leukemia" Piedrafita FJ, et al. 1996 JBC 271
14412
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Critique of a basepair
1. 97 of the genome is noncoding. 2. Even
repeats have regulatory health relevance.
3. H. sapiens as a model system Saturation
mutagenesis screen of 6x109 heterozygotes
many hits per basepair on average.
4. One key basepair may be too reductionistic.
Whole genome, whole population, whole
network analyses are becoming increasingly
feasible.
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Today's story, logic goals
Types of mutants Mutation, drift, selection
Binomial exponential dx/dt kx Association
studies c2 statistic Linked and causative
alleles Haplotypes Computing the first genome,
the second ... New technologies Random and
systematic errors
15
Where do allele frequencies come from?
Mutation (T), Migration(M), Drift (D),
Selection(S), TjSjS(SiFj-i -
SjRj-i) S(SiRi-j - SjF i-j) i0,j-1
ij1,N Mj Tj
analogous to above Dj S MiB(N,j,i/N)
i0,N Sj Dj w (wrelative fitness of i
mutants to N-i original). ________________________
__________ T,M,D,Si frequency of i mutants in a
pop. size N Fi forward rate B(N,i,PF),
Rireverse B(N,i,p) Binomial C(N,i) pi
(1-p)N-i (ref)
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Random Genetic Drift
very dependent upon population size
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Directional Stabilizing Selection

• codominant mode of selection (genic selection)
• fitness of heterozygote is the mean of the
  fitness of the two homozygotes
• AA 1 Aa 1 s aa 1 2s
• always increase frequency of one allele at
  expense of the other
• overdominant mode
• heterozygote has highest fitness
• AA 1, Aa 1 s aa 1 t
• where 0 lt s gt t
• reach equilibrium where two alleles coexist

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Fixation Times

• for neutral mutations, K µ

• for advantageous mutations, K 4Nsµ

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Role of Genetic Exchange

• Effect on distribution of fitness in the whole
  population
• Can accelerate rate of evolution at high cost
  (50)

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Network genomics
Environment
Metabolites
Interactions
RNA
DNA
Protein
Growth rate
Expression
stem cells cancer cells viruses organisms
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Multiplex Competitive Growth Experiments
t0
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64 Conditions
48 (to 600) Strains
Intensity calibrated to strain abundance (selectio
n coeficient)
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Ratio of strains over environments, e , times, te
, selection coefficients, se, R Ro
exp-?sete 80 of 34 random yeast insertions
have slt0.3 or sgt0.3 t160 generations, e1
(rich media) 50 for t15, e7. Should allow
comparisons with population allele models. Other
multiplex competitive growth experiments Thatcher
, et al. (1998) PNAS 95253. Link AJ (1994)
thesis (1997) J Bacteriol 1796228. Smith V, et
al. (1995) PNAS 926479. Shoemaker D, et al.
(1996) Nat Genet 14450.
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Today's story, logic goals
Types of mutants Mutation, drift, selection
Binomial exponential dx/dt kx Association
studies c2 statistic Linked and causative
alleles Haplotypes Computing the first genome,
the second ... New technologies Random and
systematic errors
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Caution phases of human genetics
Monogenic vs. Polygenic dichotomy Method
Problems Mendelian Linkage
need large families Common direct
(causative) 3 coding ?non-coding Common
indirect (LD) recombination new
alleles All alleles (causative)
expensive LD linkage disequilibrium
non-random association of k alleles
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Number of genes in the human genome 34,000 to
120,000
Electron magnetic moment to Bohr magneton ratio
me/mB 1.0011596521869 (41) Ur 4.1x
10-12 "99.5to accept unambiguously that the
Higgs has been spotted, the chances have to be
reduced to one in ten million" Peter J. Mohr
and Barry N. Taylor, CODATA Reviews of Modern
Physics, Vol. 72, No. 2, 2000.
physics.nist.gov/cuu/Constants Nature 407 118
Nature Genetics July 2000
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False negatives positive rates
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One form of HIV-1 Resistance
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An association test for CCR-5 HIV resistance
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But what if we test more than one locus?
The future of genetic studies of complex human
diseases. ref
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How many "new" polymorphisms?
G generations of exponential population growth
5000 N' population size 6 x 109 now N 104
pre-G m mutation rate per bp per generation
10-8 to 10-9 (ref) L diploid genome 6 x 109
bp ekG N'/N so k 0.0028 Av new
mutations lt S Lektm 4 x 103 to 4 x 104
per genome t1 to 5000
Take home "High genomic deleterious mutation
rates in hominids" accumulate over 5000
generations confound LD.
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How well linked?
G generations of exponential population growth
5000 N population size 6 x 109 now N 104
pre-G for each haplotype H, frequency of H on the
variant gametes nvH/nv frequency of H on the
gametes nH/n linkage disequilibrium d2
(nvH/nv - nH/n)2 0 to 1 q marker separation
1 recomb 1 Mbp If S sample size needed to
detect variant disease assoc. then approx. S/d2
is required for the LD marker. (Kruglyak ref)
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LD as a function of marker spacing population
expansion times
Variant at 50
Variant at 10
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LD as a function of recombination and population
size
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Finding Creating mutants
Isogenic Proof of causality Find gt Create a
copy gt Revert Caution Effects on nearby
genes Aneuploidy (ref)
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Pharmacogenomics Example
5-hydroxytryptamine transporter
Lesch KP, et al Science 1996 2741527-31
Association of anxiety-related traits with a
polymorphism in the serotonin transporter gene
regulatory region. Pubmed
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Caution phases of human genetics
Monogenic vs. Polygenic dichotomy Method
Problems Mendelian Linkage
(300bp) need large families Common
indirect/LD (106bp) recombination new
alleles Common direct (causative) 3 coding
?non-coding All alleles (109)
expensive (0.20 per SNP)
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Today's story, logic goals
Types of mutants Mutation, drift, selection
Binomial exponential dx/dt kx Association
studies c2 statistic Linked and causative
alleles Haplotypes Computing the first genome,
the second ... New technologies Random and
systematic errors
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New Genotyping

• haplotyping
  technologies
• de novo sequencing gt scanning gt selected
  sequencing gt diagnostic methods
• Sequencing by synthesis
• 1-base Fluorescent, isotopic or Mass-spec
  primer extension (Pastinen97)
• 30-base extension Pyrosequencing (Ronaghi99)
• 700-base extension, capillary arrays dideoxy
  (Tabor95, Nickerson97, Heiner98)
• SNP mapping methods
• Sequencing by hybridization on arrays (Hacia98,
  Gentalen99)
• Chemical enzymatic cleavage (Cotton98)
• SSCP, D-HPLC (Gross 99)
• Femtoliter scale reactions (105 molecules)
• 20-base restriction/ligation MPSS (Gross 99)
• 30-base fluorescent in situ amplification
  sequencing (Mitra 1999)
• Single molecule methods (not production)
• Fluorescent exonuclease (Davis91)
• Patch clamp current during ss-DNA pore transit
  (Kasianowicz96)
• Electron, STM, optical microscopy (Lagutina96,
  Lin99)

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Fluorecent primers or ddNTPs
Anal Biochem 1997 Oct 1252(1)78-88
Optimization of spectroscopic and
electrophoretic properties of energy transfer
primers. Hung SC, Mathies RA, Glazer
AN http//www.pebio.com/ab/apply/dr/dra3b1b.html
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Ewing, Hillier, Wendl, Green 1998
IndelID Total IDNS
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What are examples of random systematic errors?
For (clone) template isolation? For
sequencing? For assembly?
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Examples of systematic errors
For (clone) template isolation restriction
sites, repeats For sequencing Hairpins, tandem
repeats For assembly repeats, errors,
polymorphisms, chimeric clones, read mistracking
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Whole-genome shotgun
Project completion vs coverage redundancy
X mean coverage
(see Roach 1995)
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Weber Myers 1997
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Systematic errors
Conventional dideoxy gel with 2 hairpin
3
5
C
G
T
A
A
T
A
T A
A
Sequential dNTP addition (pyrosequencing) gt 30
base reads no hairpin artefacts
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Use of DNA Chips for SNP ID Scoring

• already used for mutation detection with HIV-1,
  BRCA1, mitochondria
• higher detection rate than gel-based assays
• higher throughput and potential for automation
• ID of gt 2000 SNPs in 2 Mb of human DNA
• can multiplex reactions

Wang et al., Science 280 (1998) 1077
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Use of Mass Spec for Analysis and Scoring
Haff and Smirnov, Genome Research 7 (1997) 378
A single nucleotide primer extension assay
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Mass Spectrometry for Analysis and Scoring
Haff and Smirnov, Genome Res. 7 (1997) 378
Use mass spec to score which base was added Can
also multiplex as long as primer masses are known
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Searching for Perls(If only finding mutations
were as easy as finding words.)
!/usr/local/bin/perl undef /
dnatext ltgt dnatext s/\gt.?\n//g
mutation text s/mutation/mutation/gi
print " found mutation\n"
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Today's story, logic goals
Types of mutants Mutation, drift, selection
Binomial exponential dx/dt kx Association
studies c2 statistic Linked and causative
alleles Haplotypes Computing the first genome,
the second ... New technologies Random and
systematic errors
52
END Sep 26, 2000
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