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Cancer Research (Fach) / V.7 Genomics/Transcriptomics (Lektion)
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- Nennen Sie den Informationsfluss des zentralen Dogma der Biologie und jeweils eine large scale Methode dazu. DNA (Genome sequencing) ->RNA (Chips/Microarrays) ->protein (Electrophoresis/Mass Spectrometry=MS) -> metabolite (Nuclear Magnetic Resonance=NMR/MS)
- Wofür steht die Abkürzung CGH? Nennen Sie die Schritte einer CGH. COMPARATIVE GENOMIC HYBRIDIZATION=CGH •Metaphase Slide Preparation•Isolation of DNA from Test Tissue and Reference Tissue•DNA Labelling•Blocking•Hybridization•Fluorescence Visualization and Imaging
- qRT-PCR: Was ist treshold cycle bzw. der Ct-Wert? Auch den Namen der gemessenen Moleküle nennen. Ct value = Cutting treshold = Difference between the starting amount of DNA, and the cycle number that you’ll reach an arbitrary number of DNA copies. = position of the lines with a value that represents the cycle number where the trace crosses an arbitrary threshold.
- Name 4 examples of SPECIFIC DETECTION SYSTEMS. -LightCycler probes (hybridisation probes) -TaqMan probes (hydrolysis probes) -Molecular beacons -Scorpion primer
- Real-time PCR instruments consist of which main components? -Thermal cycler -Optical module -PC
- What is the RIN number? The RNA integrity number (RIN) is an algorithm for assigning integrity values to RNA measurements. The RIN algorithm is applied to electrophoretic RNA measurements and based on a combination of different features that contribute information about the RNA integrity to provide a more robust universal measure. The higher the number (e.g. 10), the better the RNA sample. Unfortunately, it can´t distinguish between the different RNA origins.
- How did microarrays evolved? •One gene / one experiment–RT-PCR, Northern Blots, RNase assays–Hypothesis driven •Macroarrays–Nylon membranes–32P labelled samples •Microarrays–Glass slides–Fluorescent labelled samples–Two samples per microarray
- Definitions of -probe array -probe set -probe pair -probe cell -probe array = totality of all genes on the chip-probe set = a probe set for each gene contains 11 probe pairs (22 spots) of different probes-probe pair = contains 2 matching probe cells-probe cell = contains many copies of a specific probe, complementary to the gene probe
- What is the advantage of the Agilent Bioanalyzer over nanodrop? The results are not pH dependent and it can analyze DNA, RNA and proteins for quality and quantity.
- What is SYBR green mostly used for? As a dsDNA intercalating dye that fluoresces once bound to DNA, SYBR green is used for unspecific detection of dsDNA.
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- Difference between unspecific detection with SYBR Green and specific detection via hydrolysis and hybridization probes. SYBR Green: two primers amplify the DNA->SYBR is incorporated into the DNA->the more SYBR, the more intense the fluorescence Hydrolysis probe: primer pairs amplify DNA sequence+one additional DNA sequence/probe, that is fluorescently labeled. After amplification, the hydrolysis probe is taken away from the DNA and a fluorescence is generated, when the probe is set free. hybridization probe: primer pairs amplify DNA sequence+probe, that fluorescences, when bound to DNA. -> fluorescence is lost, when 2 dyes separate and the probe is set free.
- What is a microarray? What does is measure? Microarrays analyze gene expression by measuring mRNA. A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern. The underlying principle of microarray technology is the ability of DNA to bind to itself and to RNA. Analyzing gene expression involves the detection of mRNA species (transcriptome) present in a cell or tissue at a particular point in time.