phenotype analysis: biological-biochemical analysis · 2015-07-07 · h.schwab genetik und...
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Genetik und Gentechnik IH.Schwab
Genetic Analysis
Phenotype analysis: biological-biochemical analysis
Behaviour under specific environmental conditions
Behaviour of specific genetic configurations
Behaviour of progeny in crosses -
Genotype analysis: molecular and physical analysis
DNA Fragments/MoleculesSequence homologySequence
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Genetik und Gentechnik IH.Schwab
Genetic Analysis
Phenotype analysis: biological-biochemical analysis
Behaviour under specific environmental conditions
Some examples:
Utilization of specific substratesLoss fo function (e.g. Auxotrophy)Înduction behaviour
Behaviour of specific genetic configurations
Some examples:
Complementation analysis: e.g. function (auxotrophy) complementationCis-trans test see regulation: lac operon
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Genetik und Gentechnik IH.Schwab
Phenotype analysis: biological-biochemical analysis
Behaviour of progeny in crosses – see also introduction Mendel
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Genetik und Gentechnik IH.SchwabTetrads
Product from sexual reproduction
Specific systems is fungi
Unordered No connection to position in meiosis
Ordered Position in meiosis reflected
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Genetik und Gentechnik IH.Schwab
Ordered linear tetrads in Neurospora crassa
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
map unit (m.u.) = RF (as %)For example: 1 map unit: RF = 0,01 = 1%
1 m.u. = 1 centi Morgan (cM)
Ordered Tetrade:Distance Locus – Centromer:
(second division pattern tetrads)
Total tetradsx 1001/2 x
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Genetik und Gentechnik IH.Schwab
2 - factor cross analysis
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Genetik und Gentechnik IH.Schwab
3- factor cross analysis
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Genetik und Gentechnik IH.Schwab
13.1.15
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Genetik und Gentechnik IH.Schwab
Genetic Analysis
Genotype analysis: molecular and physical analysis
DNA Fragments/MoleculesSequence homologySequence
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab Nucleic Acid Hybridization
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Genetik und Gentechnik IH.Schwab Nucleic Acid Hybridization
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Genetik und Gentechnik IH.Schwab
Restriction site Polymorphism
markierte Sondelabelled probe
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Genetik und Gentechnik IH.Schwab
PCRPolymerase Chain Reaction
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Genetik und Gentechnik IH.Schwab
PCRPolymerase Chain Reaction
5‘
5‘
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
Short Primers Bind to many sequences
within a genomeSet of amplified fragments
depending on sequence #defined priming sites
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Genetik und Gentechnik IH.SchwabPulsed Field Gel Electrophoresis
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Genetik und Gentechnik IH.Schwab
In situ Hybridization
www.accessexcellence.org/RC/VL/GG/fish.php
FISH - (Fluoresence In Situ Hybridization)
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Genetik und Gentechnik IH.Schwab
DNA Microarrays
(DNA Chips)
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Genetik und Gentechnik IH.Schwab
DNA Sequencing
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Genetik und Gentechnik IH.Schwab
Sequencing platform
ABI3730xl Genome Analyzer
Roche (454) FLX
Illumina Genome Analyzer
ABI SOLiD
Sequencing chemistry
Automated Sanger sequencing
Pyrosequencing on solid support
Sequencing-by-synthesis with reversible terminators
Sequencing by ligation
Template amplification method
In vivo amplification via cloning
Emulsion PCR Bridge PCR Emulsion PCR
Read length 700–900 bp 200–300 bp 32–40 bp 35 bp
Sequencing throughput
0.03–0.07 Mb/h 13 Mb/h 25 Mb/h 21–28 Mb/h
www.seas.gwu.edu/~rhyspj/fall09cs144/presentations/sam.ppt
Next Generation Sequencing – Deep Sequencing
Applications: Genome sequencing, Metagenome Sequencing, Transcriptome Sequencing (incl. Non-coding RNA)Gene Expression profiling
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
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Genetik und Gentechnik IH.Schwab
Journal of Investigative Dermatology (2013), Volume 133
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Genetik und Gentechnik IH.Schwab
Sample multiplexing
http://www.illumina.com
barcode1
barcode2
Codes defined by sequence of adaptor
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Genetik und Gentechnik IH.SchwabThe Roche 454/GS FLX Sequencing Technology
Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can be amplified without contamination via the so called emulsion PCR (emPCR). The entire fragment collection is amplified in parallel.
The emPCR amplifies each fragment several million times. After amplification the emulsion shell is broken and the clonally amplified beads are ready for loading onto the fibre-optic PicoTiterDevice for sequencing
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Genetik und Gentechnik IH.Schwab Pyro-Sequencing – 454 Technology
Figure 1. Scanning electron micrograph of etched well in 454 PicoTiter sequencing plate.454's technology is based on performing hundreds of thousands of simultaneous sequencing reactions in 75 picoliter (44 μm) wells. All molecular biology reactions—DNA amplification, sequencing by synthesis, and signal light generation—occur in a single well.
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Genetik und Gentechnik IH.SchwabThe Roche 454/GS FLX Sequencing Technology
Sequencing is accomplished by synthesizing the complementary strands of the bead attached templates. In a number of cycles the four bases (ATGC) are sequentially washed over the PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic pyrophosphate starting a chemical cascade. This results in the generation of a light signal which is captured by a CCD camera.
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Genetik und Gentechnik IH.Schwab
www.illumina.com/documents/products/.../techspotlight_sequencing.pdf
DNA Sequencing with Illumina (Solexa®) Technology
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Genetik und Gentechnik IH.Schwab DNA Sequencing with Illumina (Solexa®) Technology
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Genetik und Gentechnik IH.Schwab
DNA Sequencing with Illumina (Solexa®) Technology
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Genetik und Gentechnik IH.Schwab
DNA Sequencing with Illumina (Solexa®) Technology
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Genetik und Gentechnik IH.Schwab
Ion Torrent Sequencing
http://en.wikipedia.org/wiki/Ion_semiconductor_sequencing