Co In E

MolecularToolsforGenomeAnalysis
(Chapter9:DigitalAnalysisofDNA)
Review material from 101A
Restriction enzymes to cut DNA
Gels to separate DNA/RNA by size
Probes and hybridization to specifically detect complementary
sequences
Southern blots
PCR to amplify miniscule amounts of DNA
Libraries: genomic and cDNA
DNA sequencing
233
Co In E
Sites for three restriction enzymes in
a 200 kb region of human chromosome 11
Names and location of genes in this region are shown
below the restriction sites
2
Fig. 9.3b
Co In E
Southern blots allow visualization of
rare DNA fragments in complex samples
Cut genomic DNA with restriction enzyme (s) and separate DNA
fragments by electrophoresis on agarose gel
3
Fig. 9.11
Co In E
DNA is transferred from gel to nitrocellulose membrane by blotting
DNA fragments on the membrane (blot) are in the same migration pattern
as in the gel
4
Fig. 9.11
Southern blots allow visualization of
rare DNA fragments in complex samples (cont)
Co In E
After hybridization of probe to the
blot, autoradiography reveals
fragments in restriction digests
that have sequences
complementary to the probe
5
Fig. 9.11
Southern blots allow visualization of
rare DNA fragments in complex samples (cont)
Co In E
Recombinantlibraries
Acollectionofclonesthatcontaineverysequence
fromthesourcenucleicacid
Humangenomic library:eachcloneinthelibrary
containsasinglerandomfragmentofhumanDNA
clonedinavector
cDNA library:eachclonecontainsaDNAcopyofa
singlemRNAfromthesourcetissue
237
Co In E
9.8
242
Co In E
9.9
243
Clones from a genomic library
with 20 kb inserts that are
homologous to this region
Random 100 kb genomic fragment
Clones from cDNA libraries
A comparison of genomic and cDNA libraries
Co In E
13
Please note that due to differing
operating systems, some animations
will not appear until the presentation is
viewed in Presentation Mode (Slide
Show view). You may see blank slides
in the Normal or Slide Sorter views.
All animations will appear after viewing
in Presentation Mode and playing each
animation. Most animations will require
the latest version of the Flash Player,
which is available at
http://get.adobe.com/flashplayer.
Co In E
AutomatedDNAsequencing
Fig. 9.18 a
247
Co In E
AutomatedDNASequencing
Computerreadsofthesequencecomplementarytothetemplatestrand
fromrighttoleft(5 3direction).Machinegeneratescomplementary
strand.AmbiguitiesarerecordedasanNandcansometimesbe
resolvedbyatechnician.
Fig. 9.18 c
248
Co In E
Accumulation of genome sequence data
Parallel revolutions in acquisition of genome sequence and information
technology
GenBank first official open-access, online repository for DNA sequences
(1982, National Institutes of Health)
16
Fig. 9.15a
Co In E
Ultrahigh-throughput DNA sequencing
2008 - New generation of nanotechnology-based DNA
sequencers
100 billion base pairs of sequence can be determine in a single
experiment
Millions of DNA clones can now be sequenced simultaneously
17
Fig. 9.15b
Co In E
A 3 Mb region of human chromosome 7
From human RefSeq on NCBI Sequence Viewer
Between sequence positions 116,000,001 and 119,000,000
Shows locations of nine genes, including the CFTR gene
18
Fig. 9.16b
A gene desert
Co In E
Visualization of a 540 kb region of human chromosome 7
containing the CFTR gene
From human RefSeq on NCBI Sequence Viewer
For each gene,
Exon/intron structure; blue boxes and connected lines
Spliced RNA products; red boxes
Protein coding sequences; black boxes
19
Fig. 9.16c
NCBI
Co In E
The genes for human hemoglobin are located in clusters on
two different chromosomes
The -globin (HBA) locus 28 kb on chromosome 16 contains five functional
genes (HBZ, HBM, HBA2, HBA1, HBQ1
20
The -globin (HBB) locus - 45 kb on chromosome 11 contains five functional
genes (HBE, HBG2, HBG1, HBD, HBB)
Fig. 9.19a, b
Co In E
Thesuccessionofgenesineachclustercorrelateswith
thesequenceofexpressionduringdevelopment.
globin cluster
duringthefirstfiveweeksofembryoniclife
Two chainsduringfetalandadultlife
globin cluster
duringfirstfiveweeksofembryoniclife
Two chainsduringfetallife
and withinafewmonthsofbirth
254
Co In E
Expression of Hemoglobin Gene Family Members
During Development
Fig. 9.1.c
253
Co In E
Evolutionoftheglobingenefamily
Duplicationofanancestralgenefollowedbymore
duplicationsestablishedthe andglobin
lineages.
Fig. 9.22
255
Co In E
Chapter10 Genomes
Feb 2001
256
Co In E
A comparison of the developmental complexity and
genome features of model organisms
26
Table 10.2
The information content of an organism's genome is not necessarily
proportional to its complexity
257
Co In E
HighresolutionGenomeMaps
Genomics
LinkageMaps
basedrecombinationofmolecularmarkers
PhysicalMaps
basedonrestrictionanalysisofentirechromosomes
usingoverlappinggenomiccloneswithlargeinserts
(YAC,BAC,etc)
Correlation between linkage and physical map
Humans, 1 cM~ 1 Mb; mouse, 1 cM~ 2 Mb
DNASequenceMap
258
Co In E
Molecular markers for linkage mapping
259
Co In E
Genotyping
individuals
for molecular
markers
260
Co In E
Go to live URL
262
Co In E
Fig.10.2HumanChromosomeKaryotype
263
Co In E
Fig.10.4FISHtoidentifychromosomebandthathybridizetomolecularmarkers
264
Co In E
Fig. 10.7 Spectral Karyotypes
265
Co In E
HighResolutionPhysicalMaps
Connectmolecularmarkersfromlinkage
mapstophysicalmapsofoverlapping
genomicclones
Aseriesoforderedoverlappingclonesis
calledacontig
Eventually,eachchromosomewillbe
representedbyasinglecontig.
266
Co In E
PhysicalmappingbyanalysisofSTSs
Bottomupapproach
Each STS represents a unique segment of the
genome amplified by PCR.
266a
STS = sequence tagged site
Co In E
Hierarchicalshotgunstrategy
Usedinpubliclyfundedefforttosequencehumangenome
Shear200kbBACcloneinto
~2kbfragments
Sequenceends10times
Needabout1700plasmid
insertsperBACandabout
20,000BACstocover
genome
Dataformlinkageand
physicalmapsusedto
assemblesequencemapsof
chromosomes
Significantworktocreate
librariesofeachBACand
physicallymapBACclones
Fig. 10.9
267
Co In E
Physical Map of Human Y chromosome
http://www.ncbi.nlm.nih.gov/mapview/
268
Co In E
Majorinsightsfromhumanandmodelorganism
sequences
Approximately25,000humangenes
Genesencodenoncoding RNAorproteins.
Repeatsequencesare>50%ofgenome.
Distincttypesofgeneorganization:
Genefamilies
Generichregions
Combinatorialstrategiesamplifygeneticinformationand
increasediversity.
Maleshavetwofoldhighermutationratethanfemales.
Humanraceshaveveryfewuniquedistinguishinggenes.
Alllivingorganismsevolvefromacommonancestor.
271
Co In E
Exonshuffling
Exonsoftencontaindiscreteproteindomainsor
functionalunits
ShufflingorrearrangingexoncontainingDNA
segmentscancreatenewcombinationsofprotein
domains=newproteinfunctions
Largeintronspresentalargetargetforrandom
rearrangementofthegenomewithRNAprocessing
bringingdomainstogetherinmRNA
273
Co In E
Expansion of domains and architecture of
transcription factors in specific lineages
The approximate numbers of each domain are
shown for each of the three species
40
Fig. 10.7a
274
Co In E
Geneorganizationofgenome
Genefamilies
Closelyrelatedgenesclusteredordispersed
Generichregions
Functionalorchanceevents?
Genedeserts
Span144Mbor3%ofgenome
Containregionsdifficulttoidentify?
e.g.,biggenes nucleartranscriptspans500kbor
morewithverylargeintrons(exons<1%ofDNA)
275
Co In E
Genefamilies:theolfactoryreceptorgenefamily
has1000members
276
10.11
Co In E
Phylogenetic treeofsequencerelationshipsamongORs
277
Co In E
HumanGenomeProjecthaschangedthe
potentialforpredictive/preventivemedicine.
ProvidedaccesstoDNApolymorphisms
underlyinghumanvariability
Makespossibleidentificationofgenes
predisposingtodisease
Understandingofdefectivegenesincontextof
biologicalsystems
Circumventlimitationsofdefectivegenes
Noveldrugs
Environmentalcontrols
Approachessuchasstemcelltransplantsorgene
therapy
278
Co In E
Social,ethical,andlegalissues
Privacyofgeneticinformation
Limitationsongenetictesting
PatentingofDNAsequences
Trainingofphysicians
Humangeneticengineering
Somaticgenetherapy insertingreplacement
genes
Germlinetherapy modificationsofhumangerm
line
279