II. Integrative Genomics interactions between molecules and genes

Transcription

1 . Structural Genomics Structure of Genome I. Functional Genomics Expression of Genome a. Transcriptomics b. Proteomics II. Integrative Genomics interactions between molecules and genes

2 Fields of Genomics Structural genomics Functional genomics Transcriptomics Proteomics Integrative genomics Comparative genomics Metabolomics In silico genomics Pharmacogenomics Psychogenomics

3 Structural genomics DNA sequencing In vitro DNA synthesis Determine the order of the nucleotides

4 DNA sequencing Sanger-method Template: 3 Primer : 5 CCGGTAGCAACT 5 GG 3 datp + ddatp dctp dgtp dttp datp dctp + ddctp dgtp dttp datp dctp dgtp + ddgtp dttp datp dctp dgtp dttp + ddttp GGCCA GGCCATCGTTGA GGC GGCC GGCCATC GGCCATCG GGCCATCGTTG GGCCAT GGCCATCGT GGCCATCGTT n A C G T A3 G T T G C T A C C5 Complement to the template DNA strand.

5 Automated Sanger-method

6 Large-scale, next-generation sequencing MULTIPLEX DNA SEQUENCING Hybridization-based technique SBH DNA synthesis-based method Pyrosequencing tsms SMRT Nanopore techniques

7 Sequencing by Hybridization(SBH) Genome-scale method SNP-analysis Microarray-based Not enzymatic

8 DNA synthesis-based methods of sequencing Common features DNA polymerse (enzymatic) Light detection Parallel, multiplex methods Pyrosequencing tsms: true Single Molecule Sequencing SMRT: Single Molecule Real-Time Technology

9 Pyrosequencing (DNA) n + dntp DNA polymerase (DNA) n+1 + PP i sulfurilase luciferase APS + PP i ATP light Firefly dntp ATP apirase dndp + dnmp + P i ADP + AMP + P i Pyrogram of T/G polymorphism

10 true Single Molecule Sequencing tsms Sequencing of 100 million bp single stranded DNAs at the same time Billion bases per day

11 Single Molecule Real-Time Technology SMRT 2013: 15-minute, $100 human genome

12 Nanopore sequencing Ultrafast Cheap Not enzymatic Not light detection Detection of electrict current

13 Nanopore sequencing I. Detection of a single molecule (bases) A bases of DNA pass through the nanopore, this create a change in the current Oxford Nanopore Technologies d=1nm α-hemolysin nanopore α-hemolysin nanopore + cyklodextrin

14 Nanopore sequencing II.

15 Known DNA sequences make possible the analysis of genetic diseases Identification of viruses and bacteria (causative agents) paternity test, family relationship personal identify(criminal investigation) analysis of genetic diversity investigation of new therapeutic techniques INDIVIDUAL BASED, PERSONALIZED MEDICINE

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18 Structural genomics GENOME - szekvencia megállapítása - mutációk felderítése - egy nukleotid eltérések (SNP) - deléció inszerció - metilációs mintázat DNA chips CYTOPLASM DNA Functional genomics TRANSCRIPTOM - gén expresszió megváltozása, - splice-variánsok kimutatása - szabályozó RNS-ek (mirns) kimutatása transzkripció NUCLEUS pre-mrna protein transzláció mrna ribosome trna Protein chips PROTEOM - expression - modifications - kölcsönhatások

19 A DNS chip (microarray) -It is for measuring the expression pattern of a large number of genes at the same time. One chip contains gene specific probes. There are: -cdna -oligonucleotide and -sequencing chips. Probe: affixed DNA segment

20 1. Preparing the chip: - printing - in situ synthesis control 2. Collection of tissue samples treated 3. RNA purification 4. Reverse transcription (fluorescent labeling) 5. Hybridization 6. Reading

21 In "traditional" microarrays the probes are bound to a solid surface by covalent attachment to a chemical matrix. Solid surface Glass Silicon Plastic

22 PCR - retake

23 PCR DNA-template: contains the DNA region (target) to be amplified 2 primers: complementary to the DNA regions at the 5 (five prime) and 3 (three prime) ends of the DNA region DNA-polymerase (Taq polymerase) Nucleotides: dntps, building blocks blocks from which the DNA polymerases synthesizes a new DNA strand Buffer solution: providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

24 Real-Time-PCR Used to amplify and simultaneously quantify a targeted DNA molecule Detection of fluorescence 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

25 Real-Time vs. End Point (amplification plot) real-time monitoring of DNA amplification reactions

26 RT2-PCR (reverz transcription-> Real-Time PCR) measuring mrna expression level steps RNA purification (total RNA or mrna; from cells or tissues) reverse transcription (RNA cdna) Real-Time PCR Fogalmak Real-Time PCR = qpcr (quantitative) or kinetic PCR RT2-PCR = qrt-pcr RT-PCR = reverse transcription->pcr

27 Detection Two common methods of quantification Non-specific (use of fluorescent dyes that intercalate with double-strand DNA) SYBRGreen Specific (modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA ) TaqMan probe

28 SYBR Green Intercalating dye Bind to dsdna (minor grove) It fluoresces when bound to DNA During the extension phase, more and more SYBRGreen bind to the PCR product, resulting in an increased fluorescence during each subsequent PCR cycle more fluorescence signal will be detected ssdns dsdns

29 TaqMan probe basepairs long, modified oligonucleotide 5-prime: reporter (R) dye (fluorescent label) is a fluorescent molecule that is used to monitor PCR product accumulation 3-prime: Quencher (Q) is a molecule that absorbs the energy of the reporter dye in it s excited state. Energy transfer: (FRET) Fluorescence (or Förster) Resonance Energy Transfer

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