l13 functional and_comparative_genomics
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96 97 98 99 0095
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Gen
ome
proj
ects
year
Genetics Genomics
279 completed and 543 microbial genomes in-progress
Bos taurus (cow) Canis familiaris (dog) Felis catus (cat) Homo sapiens (human) Mus musculus (mouse) Ovis aries (sheep) Pan troglodytes (chimpanzee) Rattus norvegicus (rat) Sus scrofa (pig)
Plasmodium falciparum
Danio rerio (zebrafish) Gallus gallus (chicken)
Anopheles gambiae (mosquito)Apis mellifera (honey bee) Drosophila melanogaster (fruit fly
Strongylocentrotus purpuratus (purple sea urchin)
Arabidopsis thaliana (thale cress)Avena sativa (oat)Glycine max (soybean)Hordeum vulgare (barley)Lycopersicon esculentum (tomato)Oryza sativa (rice)Triticum aestivum (wheat)Zea mays (corn)
1000 microbial genomes
4,000 genes each, 25% unique
i.e. 1.000.000 unique genes
Bible - 12,143 different words (out of 783,137)Complete works of Shakespeare - 24,000 differing words
There are fewer than 100,000 words in the French language 895,000 words in the English Language
1 gram of soil: 5,000 – 38,000 of different bacterial species
Functional genomics
Genome libraries and their screening
DNA microarrays (expression profiling)
Heterologous expression
Genome libraries and their screening
Minimal set of clones
19 klonů (E. coli DH10B pBeloBAC11)
Functional screening of genomic library
With immunized serum
DNA microarrays – regulatory gene identifications
DNA microarrays – regulatory gene identifications
6
Control Sample Treated Sample
RNA Isolation
+Cy3 + Cy5Reverse Transcription
Mix cDNAsHybridize to Array
Scan
Direct Labeling
Cloning of individual PCR products into plasmid pUNI-D-TOPO
Fusion of pUNI and pHOST plasmids
Detection of antibodies using ELISA
Statistically significant reactions of immunized serum with bacterial proteins
Statisticky signifikantní chemiluminiscenční signál
56 dní po infekci
Před infekcí56 dní po infekci
Komparative genomics
Genome sequencing
DNA microaarrays (with PCR products)
Genome fingerprints
DNA oligonucleotide microaarrays
Comparison of two bacterial genomes using DNA microaarrays containing PCR products
3113 individual hybridizations
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
r=0.98557N=3120
Log
of r
elat
ive
DN
A s
igna
l (C
unic
uli A
)
Log of relative DNA signal (Nichols)
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
N=3113r=0.98802
Log
of r
elat
ive
Cy5
sig
nal (
Nic
hol
s)
Log of relative Cy3 signal (Nichols)
DNA fingerprinting
P4
Genomic DNA
Interval A
Interval B
Primer control
Restriction digest
P1P2
P3
XL PCR
in T. pallidum subsp. pertenue Samoa D
TPI48 interval
1 – standard (1 kb)
2, 7, 12 – T. pallidum subsp. pallidum Nichols
3, 8, 13 – T. paraluiscuniculi
4, 9, 14 – T. pallidum subsp. pertenue Samoan D
5, 10, 15 – T. pallidum subsp. pallidum SS14/4934
6, 11, 16 – T. pallidum subsp. pallidum SS14/4933
• deletion 2, 5 kb u T. paraluiscuniculi
• different restriction pattern for Hind III
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
10 kb
3 kb
1 kb
EcoR I Hind III BamH I
11460 bp
DNA
Genome comparisons (NimbleGen)
29-mers (with 22 bp overlaps)
2 x 162 574 hybridizations per array
Phase II. Sequencing chip
DNA sequencing
Each nucleotide is detected by at least 8 oligos
DNA
in situ oligonucleotide synthesisNimbleGen Systems Inc.
• Digital Micromirror Device (DMD)
• Up to 386.000 features per chip