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II. GENOMICS: ANALYIS OF MULTIPLE MACROMOLECULES AT THE SAME TIME
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Genomics
Structural Functional Integrative
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Genome librariesDNA sequencingGenome projectsPolymorphismsRFLP
Genome librariesDNA sequencingGenome projectsPolymorphismsRFLP
Structural genomicsStructural genomicsStructural genomicsStructural genomics
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Construction of a human genomic DNA libraryConstruction of a human genomic DNA library
Genomic DNA library
RecombinantDNA molecules
Introduction ofplasmids into
bacteria
DNA fragmentsinserted into plasmids
Millions of genomic DNA fragments
Cleave withRestriction nuclease
Human double-stranded DNA
1.
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1 2 3 4 5 6
Constructing genome librariesConstructing genome libraries2.: ligation into plasmid vector1.: partial digestion with restriction endonuclease
1
2 53 4 6
1
2 5
3
46
2.
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3.: transforming into E. coli
3
1
2
4
5
6
3
1
2
4
5
6
Constructing genome librariesConstructing genome libraries2.: ligation into plasmid vector
3.
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Genome LibrariesGenome Libraries
Genome library: collection of clones, in wich every pieces of the genome of a particular organism can be found.
Usage: sequencing (genome projects), isolation of genes.
cDNA library: The cDNA library contains a cDNA copy of each mRNA of an organism (tissue or cell type). It represents the transcriptome.
Usage: gene structure determination, isolation of cDNSs (intronles gene).
4.
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cDNA-librariescDNA-librariesHow can we produce cDNA?
AAAAAAAAA 3’mRNA 5’
TTTTTTTTT 5’3’cDNSfirst strand
3’ CCCCC
5’ GGGGGcDNASecondstrand
3’
1. RNA (mRNA) purification: we can use total RNA or mRNA extract
2. Reverse transcription: by using of oligo dT primers and reverse transcriptase (RNA-dependent DNA polimerase) the first strand of cDNA is synthesized
3. RNase treatment
4. Linker synthesis: the terminal deoxinucleotidil transferase (DNA polimerase, which doesn’t require any template) adds the C linker to the 3’ end
5. Second strand synthesis: oligo dG primers are added and the DNA polimerasesynthesizes the second strand of cDNA.
5.
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DNA sequencing – Sanger methodDNA sequencing – Sanger methodDNA sequencing – Sanger methodDNA sequencing – Sanger method v
Dideoxynucleotide chain terminationDideoxynucleotide chain termination ororStop-nucleotide-methodStop-nucleotide-method
ororChain termination methodChain termination method
6.
Frederic Sanger
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DNA sequencing – Sanger methodDNA sequencing – Sanger method(A) Initiation of strand synthesis (B) A dideoxynucleotide
(C) Strand synthesis terminateswhen a ddNTP is added
(D) The resulting autoradiograph
A T G C DNA sequence
GAATTGGCGCGGAATTGGCGC
GAATTGGCGGAATTGGC
GAATTGGGAATTG
GAATTGAAT
GAAGA
G
Primer3’5’
3’ 5’3’5’
3’ 5’3’5’
3’ 5’
Template DNA
T T T
T T T
T T T
Base
T T T
T T T
T T T
ddA
ddA
ddA
ddAddA
ddA
The “A” family
7.
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Template: 3’
Primer : 5’
CCGGTAGCAACT 5’
GG 3’
dATP + ddATPdCTPdGTPdTTP
dATP dCTP + ddCTPdGTPdTTP
dATP dCTPdGTP + ddGTPdTTP
dATP dCTPdGTPdTTP + ddTTP
GGCCAGGCCATCGTTGA
GGCGGCCGGCCATC
GGCCATCGGGCCATCGTTG
GGCCATGGCCATCGTGGCCATCGTT
n A C G TA3’GTTGCTACC5’
Sequence complementer to the template DNA
10987654321
DNA sequencing – Sanger methodDNA sequencing – Sanger methodDNA sequencing – Sanger methodDNA sequencing – Sanger method 8.
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Automated DNA sequencing Automated DNA sequencing with fluorescently labeled dideoxynucleotideswith fluorescently labeled dideoxynucleotides
(A)
(B)
ddA
ddT
ddC
ddGddNTPs – each with a different fluorescent label
ddTddA
ddAddG
ddCddC
ddG
Sequencing reactions, fraction of products
Imaging system
Detector
Fluorescent bands move past the detector
CACCGCATCGAAATTAACTTCCAAAGTTAAGCTTGG
9.
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Craig VenterCraig Venter
Francis CollinsFrancis Collins
10.The Human Genome ProjectThe Human Genome Project
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Hierarchical method Shotgun sequencing (HGP) (Celera)Chromosomes
Fragment and sequence entire
genome
Generate and alignlarge BAC clones
Fragment and sequence a subset of the clones
11.MethodsMethods
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2 men, 3 women, 1-
1 Asian, African, Hispanic
2 Caucasian
8 Men(Unknown ethnical identity )
>50 ethnically
diverse volunteerdonors
(both sexes)(HGP)
>21ethnically
diverse volunteerdonors
(both sexes)(Celera)
2001
HGP & Celera Published a
haploid human genome
sequence
2003 First whole human genome sequence
2006 Sequence of Chromosome 1
2007 First 2 diploid genome: Venter & Watson
2008 A Han Chinese & a Yoruba men diploid genomes
12.Whose genome was sequenced?Whose genome was sequenced?
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Human Genome Project1990 – Watson, Collins, Wenter
Sequencing the whole Human Genome
Human Genome Project1990 – Watson, Collins, Wenter
Sequencing the whole Human Genome
HapMap Project2002
Mapping SNPs
HapMap Project2002
Mapping SNPs
1000 genome project2008
Sequencing the genomes of at least 1000 participant providing an overview of all genetic variations
1000 genome project2008
Sequencing the genomes of at least 1000 participant providing an overview of all genetic variations
Human Variom Project2008
Mapping the genetic variations in the Human Genome
Human Variom Project2008
Mapping the genetic variations in the Human Genome
13.Genome projectsGenome projects
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Polymorphism in Biology: having multiple alleles of a gene within a population
14.Polymorphisms/molecular markers Polymorphisms/molecular markers in Human Genomein Human Genome
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1. PCR PCR amplificationamplificationPCR primers are complementers with those DNA sequences which flank the repeats
Lenght of PCR product deppend on: Length of the „base sequence” Copy number
VNTRs: 3 person 4 pair homologous chromosomes
A B C
15.VNTR & STR analysisVNTR & STR analysis
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2. Separated by Gel Electrophoresis
Sensitive technique: it can be done from single DNA copy
DNA fingerprint
16.VNTR & STR analysisVNTR & STR analysis
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A variation in the base sequence occuring at any given single position in the genome (for example C instead of T).
ACGGCTAA
17.SNPsSNPs
It is found in more than 1% of the population.
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A variation in the base sequence occuring at any given single position in the genome (for example C instead of T).
ATGGCTAA
18.SNPsSNPs
It is found in more than 1% of the population.
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19.SNP analysis: ASASNP analysis: ASA
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RRestriction estriction FFragment ragment LLength ength PPolymorphismolymorphism
PCR primers
Restriction site mapR1 R2 R3
Polimorphic site
Agarose gelelectrophoresis
20.
PCR followedby restriction
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21.
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Microchip
Microarray scanner
22.Functional GenomicsFunctional Genomics
Real-Time PCR cycler
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GENOME TRANSCRIPTOM
PROTEOM
transcription
translation
DNS chip-ek
Protein s
- sequencing- Mutation mapping- SNPs- deletion insertion- Methylation pattern
- Alterations in gene expression,- Detection of splice variants- Detection of regulatory RNAs
- expression- Modifications- interactions
CYTOPLASM
NUCLEUS
DNA
pre-mRNS
mRNA
tRNAprotein
ribosome
Structural genomics Functional genomics
Chip (microarray) technologyChip (microarray) technology 23.
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DNA chipDNA chipDNA chipDNA chip
It is for measuring the expreesion pattern of a large number of genes at the same time
A chip contains 6-10000 gene specific probes
There are cDNA & oligonucleotide microarrays
24.
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1. Preparing the chip: - printing
(in situ synthesis)
2. Collection of tissue samples
control disease
3. RNA purification
4. Reverse transcription (fluorescently labeling)
5. Hybridization 6. Reading
25.
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Outline of a microarray analysis
3. Scan the chip
2. Hybridize labeled probe withDNA microarray on a chip
1. Isolate RNA samples. Synthesize DNA copies. RNA population or probes
Clinical sample
4. Analyze data andcorrelate with
histoclinical data
8x4x2 2x4x8
26.
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Treat cells with formaldehydeSonicate to produce fragmentsof chromatin
Reverse cross linksPurify DNA
DNA fragment containing specifictranscription factor binding site
Collect chromatin-antibodycomplex
Antibody bonds specifictranscription factor
Fragments of chromatin withtranscription factorscross-linked to DNA
Immunoprecipitatewith antibody
ChIP (Chromatin immunoprecipitation)ChIP (Chromatin immunoprecipitation) 27.
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Real-Time-PCRReal-Time-PCR28.
Used to amplify and simultaneously quantify a targeted DNA molecule
Detection of fluoresce at each cycle during PCR reaction → Real-Time
No gel-based analysis at the end of the PCR reaction
Computer based analysis of the cycle fluorescence time course
Real-Time PCR cycler
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Real-TimeReal-Time vs. vs. End PointEnd Point29.
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• Measuring mRNA expression level• „steps”:
– RNA purification– Reverse transcription– (RNA cDNA)– Real-Time PCR
• „Terms”• Real-Time PCR = qPCR (quantitative)• RT2-PCR = qRT-PCR• RT-PCR = reverse transcription followed by PCR
30.RT2 PCRRT2 PCRRT2 PCRRT2 PCR
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33.Real-Time PCR – measuring expressionReal-Time PCR – measuring expressionReal-Time PCR – measuring expressionReal-Time PCR – measuring expression
• Treated – untreated samples• Healthy – diseased (eg. Tumour and normal tissue samples)
• More sensitive than microarray• Less samples at a time• Measuring relative copy number
(∆Ct)
31.
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Met Met
Sodium-bisulfiteSodium-bisulfite CpG CpGCpG CpG
CpG UpGCpG UpG
1. Treatment with Sodium-bisulfite
2a MethylC-seq
2b Real-Time PCR
DNA methylation analysisDNA methylation analysis32.
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DNA methylation analysis– MethylC-seqDNA methylation analysis– MethylC-seq
Genomic DNA
Random fragmentation
33.
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DNA methylation analysis– MethylC-seqDNA methylation analysis– MethylC-seq
Non-methylated CMethylated C
34.
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DNA methylation analysis - Real-Time PCRDNA methylation analysis - Real-Time PCR
Genomic DNA
Bisulfite conversion
Real-Time PCR
35.