6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interest in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
GJ461DS467
6.1 Positional cloning – genetic and physical map 2
GJ17
GJ16
GJ162
GJ131
DS71GJ476
GJ461
GJ10GJ01
DS09
DS467
GJ367
GJ402
GJ403
GJ162 GJ10
GJ70GJ21
GJ18
GJ392 GJ511GJ367
GJ29
GJ44
GJ531
0.21.8 0.2 0.40.3 0.1 0.6 0.2 8.4SP6
T7
cM
3.8 cM
6.1 Positional cloning – Map Based Cloning 3
Ai l i f fPositional Cloning of the Hs1pro-1 Gens
• Aim: cloning of a gene of interest with an unknown gene product
1: Marker-IsolationHs1pro-1
• Method: Localisation of gene
1: Marker-Isolation
2: YAC/BAC-contig
Tl: 940043
• Method: Localisation of gene position by
1 genetic mapping 3: cDNA-Isolation1. genetic mapping2. physical mapping3. cloning of the interesting
i ( i )4: Genetic
Hs1pro-1
region (contig)4. Searching candidate
genes
complementation
6.1 Positional cloning – minimal tiling path 4
Hs1pro-1 A5-117-T7O6-100-T7 23a + 14b 128R 104RA906001
Pro4
A906001
A19-62 115kb
K15-57 120kbK15 57 120kb
O5-132 140kb
I15 137I15-137 145kb
G11-123 140kb
B.procumbens specific probe
i l
A23-67 130kb
T7-End Sp6-EndBAC-end-sequence
signal on BAC
6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interest in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
6.2 Insertion mutagenesis - Transposon tagging 6
- Two elements: Ac / Ds:
Ac Activator1 2 copies per Cell1-2 copies per Cellaktive transposonconfers production of transposase
Ds Dissociation5-100 copies per Cell5 00 cop es pe Ceinaktive transposon (deleted Ac-element)no transposasecan be trans-activated by the Ac-element
6.2 Insertion mutagenesis - Transposon tagging 7
Phenotype Ie
(resistent) Cloning of the candidate geneg
e
Phenotyp II(susceptibel) no protein - MUTATION
mRNA disruption
Production of a mutant populationi f i t ti h t
7screening for interesting phenotypes
6.2 Insertion mutagenesis – inverse PCR 8
known sequence (e.g. transposon) UNKNOWN sequence
EcoRI EcoRI EcoRIEcoRI
Restriction
EcoRI EcoRI EcoRIEcoRI
PrimerdesignEcoRI EcoRIf
r
f
iPCR fragment
Ligationr
f
PCRelektrophoreses, cloning r
6.2 Insertion mutagenesis - T-DNA tagging 9
GENE X
Wild type = X+
T-DNAE XGENE X
Arbitrary primer (ap)
Nested specific primers1
23
PCR 1 with ap + 1(low stringency)
Mutant = x-
( g y)
PCR 2 with ap + 2
1 2 3
PCR 3 with ap + 3
6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interset in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
6.3 PCR-cloning by use of homologue sequence 11
Amino acid sequences are highly conserved
X 21 (Ri X h )
Pto (Tomato, Pseudomonas )
Ser/Thr-Protein Kinase
N (Tabacco, TMV)
Xa21 (Rice, Xanthomonas)
Cf9 (Tomato, Cladosporium )
Cre3 (Wheat, Heterodera )
Prf (Tomato, Pseudomonas ) LZ: Leucine-ZipperNBS: Nucleotide-Binding SiteLRR: Leucin-rich RegionTMD: Trans Membrane Domaine
Gpa2 (Potato, Globodera)
Mi (Tomato, Meloidogyne )
TMD: Trans Membrane DomaineTIR: Toll/Interleukin-1 Signal domaine
Hero (Tomato, Globodera)
Gro1-4 (Potato, Globodera)
Hs1pro-1(sugar beet, Heterodera )
LRRLZ/CC NBS TMD
6.3 PCR-cloning by use of homologue sequence 12
Resistance gene analogues (RGA) in Arabidopsis
6.3 PCR-cloning by use of homologue sequence 13
R P-loop kinase-2 kinase-3a GLPL TIR/CC
Prf GLGKTTLAKKIY KRFLILIDDVW SNRSRIILTTR RGLPLSVVLV LKPCFLYFGGFL
Cre3 GSGKSTLAQFVY KRFLLVLDDVW KKGSKILVTTR KGSPLAARTV ARRCFAYCSIFP
Mi GSGKTTLAYKVY KRYLIVLDDVW KKGSRIILTTR KGLPLVADLI LKPCLLYFASFPMi GSGKTTLAYKVY KRYLIVLDDVW KKGSRIILTTR KGLPLVADLI LKPCLLYFASFP
Gpa2 GIGKTTLAAKLY RRYLVVIDDIW DNGSRILLTTR GGLPLAITLI LKPCFLYFAIFA
Hero GVGKTTLANKVY KRYLIVLDDVW ERGNRVILTSR KGLPLALVLI LKPLLLYFARLQ
Cons GSGKTTLA KRYLIVLDDVW KKGSRILLTTR KGLPLAAVLI LKPCFLYFAIFP
R-s1 R-as1
R-s1: G K T T L A
Design of degenerated primer
5´- GGN AAR ACN ACN YTN GC (deg.)
R-as1: K G L P L A5´- GC NAR NGG NAR NCC YTT (deg.)5 GC NAR NGG NAR NCC YTT (deg.)
H=A/T/C; R=A/G; N=G/A/T/C; s=sense; as=antisense
6.3 PCR-cloning by use of homologue sequence 14
6.3 PCR with degenerated primer 15
Using the IUB-codes (International Union of Biochemistry)IUB Code
N (I) V B H D K S W M Y R
Basen A,C,G,T G,A,C G,T,C A,T,C G,A,T G,T G,C A, T A,C C,T A,G
Leucine Lysine Proline Cysteine Phenylalanine Leucine Tyrosine Alanine Isoleucine Phenylalanine Alanine
AA Leu Lys Pro Cys Phe Leu Tyr Ala Ile Phe Ala
Basen CAC A
AC C C
AC
AA C
AC
TT
GT
A AG
C C CGT
T GT
T TT T
T CGT
T AT
G C CGT
A TCT
T TT
G CGT
IUB Y T N A A R C C N T G Y T T Y Y T N T A Y G C N A T H T T Y G C N
6.3 PCR-cloning by use of homologue sequence 16
Q L N A T L N HCAG CTG AAC GCG ACG CTG AAC CACCAA CTC AAT GCC ACC CTC AAT CAT
AS-Sequenz
CAA CTC AAT GCC ACC CTC AAT CATCTA GCA ACA CTACTT GCT ACT CTTTTG TTG
reverse TranslationTTG TTGTTA TTA
C G G G G3‘-GT AI TT CGI TGI AI TT GT-5‘
T A A A A Primer-SequenzT A A A A q
PCRPCR
Cloning and sequencing
H=A/T/C; R=A/G; I=G/A/T/C; s=sense; as=antisense
6.3 PCR amplification with degenerated primer 17
r s w r s w 4
Degenerated DegeneratedDegenerated Primer pair: R3/R6
DegeneratedPrimer pair: Gpa2s1/as1
6.3 PCR amplification with degenerated primer 18
Z-1
Mi, Prf, RPM1Z-3
Mi, Prf, Gpa2, Rx
Z-7
Mi, Prf, Gpa2, Rx
Mi, Prf, Gpa2, Rx
Z-9
Mi P f RPM1Mi, Prf, RPM1
6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interest in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
6.4 Cloning of genes of interest in silico 20
Sequencing of largegenome regions g g
looking for ORFslooking for ORFs
Database searchAnnotation
? ?
Selection of did tcandidate genes
FunctionalFunctionalcharacterization
6. Cloning of genes of interes6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interst in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
7.1 RNA, mRNA and cDNA 22
Genome
genomic DNA, gDNAR bi ti d t tiRecombination and mutation
Transkriptomep
mRNA, ESTs, cDNAEnvironment
Proteome
structural proteins, enzymesp , yEnvironment
Metabolome
Metabolites, secondary metabolitesEnvironment
7.1 RNA, mRNA and cDNA 23
cDNA-synthesis
5´ 3´AAAAAAA mRNA<TTTTTTTT 5´
i cDNAR primer cDNAfirst strand synthesis
ReverseTranscriptase (RT) = RNA dependent DNA-polymerase
Degradation of the RNA + <AAAAAAA 3´5´ second strand synthesis<TTTTTTTT 5´<AAAAAAA 3
3´5
Cloning (Plasmid vector)
Sequencing
6. Cloning of genes of interes6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interst in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
257.2 EST/cDNA libraries
EST = Expressed Sequence Tags
Gene expression
depending on organs, developmental stage and environment
267.2 EST/cDNA libraries - EST assembling
ESTs = roughly sequenced transcripts (J. C. Venter)
tissue (e.g. roots)
RNA-preparation
cDNA synthesiscDNA synthesis
cDNA-clone library
random sequencing
high number of clones
t lgene catalog
7.2 EST/cDNA libraries 27
Construction of a cDNA-library
Phage library
odod.
7.2 EST/cDNA libraries 28
Construction of a cDNA-library
Plasmid-libraryAdaptor ligation
<GAGCTCTTTTTTTT 5´<CTCGAGAAAAAAA 3´
3´ CTTAAG5´ GAATTC
Adaptor ligation
EcoRI XhoI
EcoRI
Xh IXhoI
7.2 EST/cDNA libraries - screening 29
7.2 EST/cDNA libraries - screening 30
screen I screen II
317.2 EST/cDNA libraries
• ESTs are an evidence for transcription of a certain sequence
• ESTs are tools to identify new genes (dbEST-search)
• The relative amount of EST-sequences is proportional to its expression of a gene in one experiment > Possibility of a virtual northern
b t 60 i EST kabout 60 mio. EST-sequences are known
more than 10 mio. ESTs from 200 plant species, ordered by tissue, culture conditions etcculture conditions etc.
Species Family No. of ESTs
327.2 EST/cDNA libraries - EST assembling
7.2 EST/cDNA libraries - RACE 33
Region of known EST sequence
FROM EST TO VALID mRNA-SEQUENCE
Region of known EST-sequence
Region to be amplifiedby 5´-RACE
Region to be amplifiedby 3´-RACE
GSP2 NGSP2 NGSP1 GSP1Universalprimer
Universalprimer
NNAAAA-3NNTTTTT-5´_UPBS
UPBS_5´3´
RACE = Rapid Amplification of cDNA-Ends
6. Cloning of genes of interes6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interst in silico
7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies
7.3 SSH, Genechips and RNAseq technologies 35
Technologies of Transcriptome-analysis
Question: Which genes are involved in a certain trait?Question: Which genes are involved in a certain trait?
Possibility of identification of up- and down-regulated genes(i ti d ifi i t l diti / t t t )(in a tissue, under specific environmental conditions / treatments.....)
Expression profiling
Finding candidate genes for the trait (phenotype)
With sequence information
Withoutsequence informationq q
SSH ( )RNAseq
Genechips
7.3 Construction of an SSH-library 36
Resistant plants20 plants 20 plants 20 plants
3 DAI 6 DAI 12 DAI
Susceptible plants20 plants 20 plants 20 plants
3 DAI 6 DAI 12 DAI3 DAI 6 DAI 12 DAI 3 DAI 6 DAI 12 DAI
RNA-IsolationRNA RNA RNA RNA RNA RNA
pooling
RNA RNA RNA RNA RNA RNA
mRNA-RTester / DriverForward-SubtractionR-Gene
mRNA-S
Driver / TesterReverse-SubtractionForward Library Reverse Library
up-regulated genes down-regulated genes
Aim: Identification of genes related to R-gene mediated plant-Resistance
7.3 Principle of SSH-libraries 37
SSH- Subtraktive Supression Hybridisation
Adaptor-Ligation (F/R)
1. Hybridization
2. Hybridization
Amplification Cloning Sequencing
7.3 SSH library 38
No special equipment necessary (only sequencing instrument)☺ p q p y ( y q g )
S i bl f k d ( i i f )
☺
☺ Suitable for weak expressed genes (e.g. transcription factors)☺
Amplification of long cDNA-molecules (easy to annotate)☺
Analysis of known and unknown transcripts☺
One SSH-procedure for each comparison, time-point, tissue
7.3 Genechips (Microarrays) 39
1. Thousands of defined oligonukleotides are immobilised on an glass-array (chip)
2. Each oligonucleotide represents a transcript
3. Hybridisation with fluorescence-labeled cDNA from different tissues, environments, treatmrents...
Oligonucleotides as probes at a
labeled cDNA-molecules
Hybridisation with the complementary oligonucleotideas probes at a
specific positionon the array
oligonucleotide
7.3 Genechips (Microarrays) – differential hybridization 40
7.3 Genechips (Microarrays) – differential hybridization 41
7.3 Genechips (Microarrays) – differential hybridization 42
upregulated in treatment A
upregulated in treatment B(= downregulated in treatment A)
no expression in A and B
equal expression in A and B
7.3 Genechips (Microarrays) 43
Special equipment necessary (expensive)
Only known genes can be analysed
Weak and strong expressed genes can be analysed☺
> 100 Arrays per production process☺high number of quick and easy experimentshigh number of quick and easy experimentsonly RNA-isolation, labeling and hybridization
7.3 RNAseq – trancriptome sequencing 44
New generation sequencing of cDNA libraries(454 Illumina )(454, Illumina....)
Quantitative analysis of gene expression Q y g pby counting reads per transcript
7.3 RNAseq – trancriptome sequencing 45
Sanger Roche454
IlluminaHiSeq2000g 454 HiSeq2000
Read length: 1000 bp 400 bp 100 bp
Sequenz-informationper run
0,0003 GBp 0,6 GBp > 200 GBpper run
7.3 SSH, Genechips and RNAseq technologies 46
After performing an expression profiling experiment
1 I ili l i l ti f i t ti did t1. In silico analysis selection of interesting candidate-genesfrom a huge ammount of data
2. Verification of expression profile results by an independentexperimental system
3. Functional characterization of candidate genes
7.3 SSH, Genechips and RNAseq technologies 47
1. In silico analysis selection of interesting candidate-genesfrom a huge ammount of data
1000
TreatmentsA B C D
Trea
tmen
t A
10
100
1 10 100 1000
1
Genetic Characteristic WT (raw)
Treatment B
Cell rescue, defense,cell death and ageing
16% Cellular communication/
Cell growth, celldivision and DNA synthesis
2%16% Cellular communication/
signal transduction
5%
Cellular organization2%
Cellular transport andtransport mechanisms
Unknown34%
6
p4%
Energy4%Transcription
5%
Protein-Synthese14%
Protein-Destination4%
Metabolism10%
7.3 SSH, Genechips and RNAseq technologies 48
2. Verification of expression profile results by an independentexperimental system
qPCR N th
Gene 1
qPCR Northern
Gene 2
Gene 1
Gene 1 Gene 2 PR2 Gen 5 Gen 6Gene 3 Gen 7Gene 4 HK
Gene 4
Gene 3
Gene 1 Gene 2 PR2 Gen 5 Gen 6Gene 3 Gen 7Gene 4 HK
7.3 SSH, Genechips and RNAseq technologies 49
3. Functional characterization of candidate genes
1. Genetic complementation
2. Knock out mutants / RNAi2. Knock out mutants / RNAi
3. Protein function / protein assays
4. Promoter analysis
G HG H
Thank you for your attention.