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

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

2. Genomics Structural Functional Integrative

3. Structural genomics • Genome libraries • DNA sequencing • Genome projects • Polymorphisms • RFLP

4. 1. Construction of a human genomic DNA library Cleave with Restriction nuclease Human double-stranded DNA Millions of genomic DNA fragments DNA fragments inserted into plasmids Introduction of plasmids into bacteria Recombinant DNA molecules Genomic DNA library

5. 2. Constructing genome libraries 2.: ligation into plasmid vector 1.: partial digestion with restriction endonuclease 2 4 3 6 5 1 5 2 3 1 2 3 4 5 6 1 4 6

6. 3. Constructing genome libraries 5 2 2.: ligation into plasmid vector 3.: transforming into E. coli 4 3 1 6 5 2 3 1 4 6

7. 4. Genome 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).

8. 5. cDNA-libraries How can we produce cDNA? 3’ cDNA Second strand mRNA AAAAAAAAA 3’ 5’ GGGGG 5’ cDNS first strand 3’ 3’ CCCCC TTTTTTTTT 5’ • 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:theterminal 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 polimerase • synthesizes the second strand of cDNA.

9. v Dideoxynucleotide chain termination or Stop-nucleotide-method or Chain termination method 6. DNA sequencing – Sanger method Frederic Sanger

10. 7. DNA sequencing – Sanger method (A) Initiation of strand synthesis (B) A dideoxynucleotide Primer Template DNA 5’ 3’ T T T 3’ 5’ Base 5’ 3’ T T T 3’ 5’ 5’ 3’ T T T 3’ 5’ (D) The resulting autoradiograph (C) Strand synthesis terminates when a ddNTP is added ddA DNA sequence A T G C T T T ddA GAATTGGCGCG GAATTGGCGC T T T GAATTGGCG ddA GAATTGGC GAATTGG T T T GAATTG GAATT ddA GAAT GAA ddA GA G ddA The “A” family

11. DNA sequencing – Sanger method 8. Template: 3’ CCGGTAGCAACT 5’ Primer : 5’ GG 3’ dATP dCTP + ddCTPdGTPdTTP dATP dCTPdGTP + ddGTPdTTP dATP + ddATPdCTPdGTPdTTP dATP dCTPdGTPdTTP + ddTTP GGCCATGGCCATCGTGGCCATCGTT GGCCAGGCCATCGTTGA GGCGGCCGGCCATC GGCCATCGGGCCATCGTTG n ACGT A3’GTTGCTACC5’ 10987654321 Sequence complementer to the template DNA

12. Automated DNA sequencing with fluorescently labeled dideoxynucleotides 9. (A) ddA ddC ddNTPs – each with a different fluorescent label ddT ddG Sequencing reactions, fraction of products ddT Imaging system ddA ddA ddG ddC Detector ddC ddG Fluorescent bands move past the detector (B) CACCGCATCGAAATTAACTTCCAAAGTTAAGCTTGG

13. 10. The Human Genome Project Craig Venter Francis Collins

14. 11. Methods Hierarchical method Shotgun sequencing (HGP) (Celera) Chromosomes Generate and alignlarge BAC clones Fragment and sequence entire genome Fragment and sequence a subset of the clones

15. 12. Whose genome was sequenced? >21 ethnically diverse volunteerdonors(both sexes) (Celera) >50 ethnically diverse volunteerdonors(both sexes) (HGP) 2001 HGP & Celera Published a haploid human genome sequence 2 men, 3 women, 1-1 Asian, African, Hispanic 2 Caucasian 8 Men (Unknown ethnical identity ) 2003 First whole human genome sequence 2006 Sequence of Chromosome 1 2007 First 2 diploid genome: Venter & Watson 2008 AHan Chinese & a Yoruba men diploid genomes

16. Genome projects 13. Human Genome Project1990 – Watson, Collins, WenterSequencing the whole Human Genome HapMap Project2002Mapping SNPs 1000 genome project2008Sequencing the genomes of at least 1000 participant providing an overview of all genetic variations Human Variom Project2008Mapping the genetic variations in the Human Genome

17. Polymorphisms/molecular markers in Human Genome 14. Polymorphism in Biology: having multiple alleles of a gene within a population

18. VNTR & STR analysis 15. • 1. PCR amplification • PCR 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

19. 16. VNTR & STR analysis 2. Separated by Gel Electrophoresis Sensitive technique: it can be done from single DNA copy DNA fingerprint

20. SNPs 17. A variation in the base sequence occuring at any given single position in the genome (for example C instead of T). ACGGCTAA It is found in more than 1% of the population.

21. SNPs 18. A variation in the base sequence occuring at any given single position in the genome (for example C instead of T). ATGGCTAA It is found in more than 1% of the population.

22. 19. SNP analysis: ASA

23. RFLP 20. Restriction Fragment Length Polymorphism Polimorphic site R1 R2 R3 Restriction site map PCR primers Agarose gel electrophoresis PCR followed by restriction

24. 21. RFLP

25. Functional Genomics 22. Microchip Microarray scanner Real-Time PCR cycler

26. 23. Chip (microarray) technology Structural genomics Functional genomics DNS chip-ek GENOME TRANSCRIPTOM - sequencing - Mutation mapping - SNPs - deletion  insertion - Methylation pattern - Alterations in gene expression, - Detection of splice variants - Detection of regulatory RNAs CYTOPLASM DNA NUCLEUS transcription Protein s pre-mRNS tRNA protein translation PROTEOM - expression - Modifications - interactions mRNA ribosome

27. 24. DNA 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

28. 25. • 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

29. 26. Outline of a microarray analysis 1. Isolate RNA samples. Synthesize DNA copies. RNA population or probes Clinical sample 4. Analyze data and correlate with histoclinical data 2. Hybridize labeled probe with DNA microarray on a chip 2x4x8 8x4x2 3. Scan the chip

30. ChIP (Chromatin immunoprecipitation) 27. Antibody bonds specific transcription factor Collect chromatin-antibody complex Immunoprecipitate with antibody Treat cells with formaldehyde Sonicate to produce fragments of chromatin Reverse cross links Purify DNA DNA fragment containing specific transcription factor binding site Fragments of chromatin with transcription factors cross-linked to DNA

31. Real-Time-PCR 28. • 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

32. Real-Time vs. End Point 29.

33. Measuring mRNA expression level „steps”: RNA purification Reverse transcription (RNA cDNA) Real-Time PCR 30. RT2 PCR • „Terms” • Real-Time PCR = qPCR (quantitative) • RT2-PCR = qRT-PCR • RT-PCR = reverse transcription followed by PCR

34. 31. 33. Real-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)

35. 32. DNA methylation analysis • 1. Treatment with Sodium-bisulfite • 2a MethylC-seq • 2b Real-Time PCR Met Met Sodium-bisulfite CpG CpG CpG UpG

36. 33. DNA methylation analysis– MethylC-seq Genomic DNA Random fragmentation

37. 34. DNA methylation analysis– MethylC-seq Non-methylated C Methylated C

38. 35. DNA methylation analysis - Real-Time PCR Genomic DNA Bisulfite conversion Real-Time PCR