l13 functional and_comparative_genomics

96 97 98 99 0095

20

40

60

80

100

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