II. GENOMICS: ANALYIS OF MULTIPLE MACROMOLECULES AT THE SAME TIME

Genomics

Structural Functional Integrative

Genome librariesDNA sequencingGenome projectsPolymorphismsRFLP

Genome librariesDNA sequencingGenome projectsPolymorphismsRFLP

Structural genomicsStructural genomicsStructural genomicsStructural genomics

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.

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.

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.

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.

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.

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

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.

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.

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.

Craig VenterCraig Venter

Francis CollinsFrancis Collins

10.The Human Genome ProjectThe Human Genome Project

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

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?

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

Polymorphism in Biology: having multiple alleles of a gene within a population

14.Polymorphisms/molecular markers Polymorphisms/molecular markers in Human Genomein Human Genome

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

2. Separated by Gel Electrophoresis

Sensitive technique: it can be done from single DNA copy

DNA fingerprint

16.VNTR & STR analysisVNTR & STR analysis

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.

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.

19.SNP analysis: ASASNP analysis: ASA

RRestriction estriction FFragment ragment LLength ength PPolymorphismolymorphism

PCR primers

Restriction site mapR1 R2 R3

Polimorphic site

Agarose gelelectrophoresis

20.

PCR followedby restriction

21.

Microchip

Microarray scanner

22.Functional GenomicsFunctional Genomics

Real-Time PCR cycler

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.

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.

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.

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.

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.

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

Real-TimeReal-Time vs. vs. End PointEnd Point29.

• 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

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.

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.

DNA methylation analysis– MethylC-seqDNA methylation analysis– MethylC-seq

Genomic DNA

Random fragmentation

33.

DNA methylation analysis– MethylC-seqDNA methylation analysis– MethylC-seq

Non-methylated CMethylated C

34.

DNA methylation analysis - Real-Time PCRDNA methylation analysis - Real-Time PCR

Genomic DNA

Bisulfite conversion

Real-Time PCR

35.