einführung in die genetik - plantdev.wzw.tum.de
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Einführung in die Genetik
Prof. Dr. Kay Schneitz (EBio Pflanzen)http://[email protected]
Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/[email protected]
Einführung in die Genetik - InhalteEinführung in die Genetik - InhalteEinführung in die Genetik - Inhalte1 Einführung 15. 10. 13 KS2 Struktur von Genen und Chromosomen 22. 10. 13 KS3 Genfunktion 29. 10. 13 KS4 Transmission der DNA während der Zellteilung 05. 11. 13 KS5 Vererbung von Einzelgenveränderungen 12. 11. 13 KS6 Genetische Rekombination (Eukaryonten) 19. 11. 13 KS7 Genetische Rekombination (Bakterien/Viren) 26. 11. 13 KS8 Rekombinante DNA-Technologie 03. 12. 13 CS9 Kartierung/Charakterisierung ganzer Genome 10. 12. 13 CS
10 Genmutationen: Ursache und Reparatur 17. 12. 13 CS11 Veränderungen der Chromosomen 07. 01. 14 CS12 Genetische Analyse biologischer Prozesse 14. 01. 14 CS13 Transposons bei Eukaryonten 21. 01. 14 CS14 Regulation der Zellzahl 28. 01. 14 CS15 Regulation der Genexpression 04. 02. 14 KS
Recombinant DNA Technology
Genetics 08
Based on Chapter 11 (Griffiths; 10th ed.)
Summary • Plasmids and vectors
• origin of replication (ori)
• selection markers (AmpR, TetR etc.)
• polylinker = multiple cloning site
• restriction sites
• blue-white selection (LacZ)
• Cloning, recombination technology, genetic engineering
• Restriction enzymes
• sticky and blunt ends
• 5’ overhangs, 3’ overhangs
• methylation of DNA in E. coli
• T4 Ligases and ligation
• Topoisomerase-based cloning
• Recombination-based cloning (e.g. Gateway system)
• Polymerase chain reaction
• Melting, annealing, extension
• Taq polymerase
• primer, oligo(-nucleotide)
• Transformation of ligation product to E. coli
• Heat shock
• Electroshock
• DNA Preparation using alkaline lysis
• DNA sequencing
• Sanger sequencing (dideoxy sequencing, chain termination sequencing)
• Next generation sequencing
• Third generation sequencing
Summary
E.coli strains (examples)
XL1 Blue - for cloning
Rosetta(DE3)pLysS - for protein expression
Mapping and characterization of entire genomes
Genetics 09
Based on Chapter 15 (Griffiths; 10th ed.)
How are genome sequences obtained?
How is this information deciphered?
How can comparing genomes help to understand life and evolution?
The advances through next generation sequencing
How does the availability of genome sequences affect biological analyses?
How are genome sequences obtained?
Restriction enzymes
Digest of genomic DNAM size marker
1,3 undigested
2,4 digested
Digest of plasmid DNAM size marker
1,3 undigested
2,4 digested
Agarose gel Agarose gel
Genome sequencing strategy
Arabidopsis125 000 000 bp
Large fragmentsx 100 000 bp
Orderd large fragments(minimal tiling path)
Make small fragments from large fragmentsx 500 - 1000 bp
Sequence, align and overlap reads (contig)
Sequencing length 500-1000 bp/run
Assemble chromosomes and genome
Vectors for large inserts
BACs100 - 300 kb
YACs50 - 2000 kb
Phage Lambda35 - 45 kb
BAC, bacterial artificial chromosomeYAC, yeast artificial chromosome
Vectors for small inserts
Plasmid vectors<10-15 kb maximum
ca. 500 - 1000 bp inserts for sequencing
Genome sequencing strategyArabidopsis125 000 000 bp
Large fragmentsx 100 000 bp
Orderd large fragments(minimal tiling path)
Make small fragments from large fragmentsx 500 - 1000 bp
Sequence, align and overlap reads (contig)
Assemble chromosomes and genome
Generating physical maps
Generating a minimal tiling path
From paired end reads to a contig
Filling contig gaps
How is genome sequence deciphered?
Genome size
Bacteriophage fx 174 (5.3 kb, first sequenced genome 1977)Mitochrondrial DNA (human; 16.3 kb)Bacteriophage l (48.5 kb)Chloroplast DNA (Marchantia; 121 kb)Vaccinia virus (192 kb)Cytomegalovirus (CMV; 229 kb)Bacteria (Haemophilus influenzae; 1,830 kb)Bacteria (Escherichia coli; 4,600 kb)Yeasts (Saccharomyces cerevisiae; 12,100 kb)Insects (Drosophila; 130,000 kb)Plant (Arabidopsis; 157,000 kb)Man (3,200,000 kb)Plant (Wheat; 17,000,000 kb)Fish (Protopteros aethiopicus; 130,000,000 kb) = largest genome
Chromosome numbers
Segmentally duplicated regions in the Arabidopsis genome. The Arabidopsis genome initiative, Nature 408, 796-815 (2000)
DNA sequence comparison
SyntenyArabidopsis ChromosomeNOR, nucleolus organizing region
Elements and sites be recognized by more or less conserved DNA sequence elements, can therefore be predicted by bioinformatics
Exon/intron structure particularly important because it allows to predict the sequence of a protein
Structure of a eukaryotic gene
cDNA = complementary DNA of mRNA
EST = expressed sequence tag, sequenced cloned mRNA/cDNA
Predicting and confirming genesfrom a genomic sequence and cDNA/ESTs
Translating genomic information into protein
Making gene predictions based on genome sequence
No correlation between genome size and gene numbers
Number of genes Genome size (Mb)
How comparing genomes can help to understand life and evolution
Segmentally duplicated regions in the Arabidopsis genome. The Arabidopsis genome initiative, Nature 408, 796-815 (2000)
Synteny
SyntenyArabidopsis ChromosomeNOR, nucleolus organizing region
Genome evolution
Synteny
The advances through next generation sequencing
Next generation sequencing
Shearing of the DNA!to 300 - 800 bp fragments!
Adaptor ligation!B Primer is biotinylated!
Streptavidin beads capture biotinylated B primed fragments -> Emulsion PCR is used to amplify fragment on the beads!
Distribution of beads to a fibre-optic PicoTiterDevice!
Million fold amplification of PCR!fragment on the beads!
Pyrosequencing!
Roche 454/GS FLX Sequencing Technology
N.b. next generation sequencing allows to obtain full genome sequences while omitting the cloning steps, thus saving time and cost.
An assembled and related genome may be used as a scaffold for genome assembly
The information about the full genome architecture may not be required in
A genome sequence map
Genome sequencing is automated
...and largely institutionalized
NGS genome sequencing revolution
• (Crop) plant genomes (published) rice (2002) poplar (2006) grape (2007) papaya (2008) cucumber (2009) maize (2009) sorghum (2009) soybean (2010) apple (2010) strawberry (2010)
• Model plant genomes Arabidopsis (2000) Brachypodium (2010) 1001 Genomes project (2011)
The 1000 (Human) Genomes project
How does the availability of genome sequences affect biological analyses?
Functional studies - Gene knock outs
Functional studies - Gene targeting
Functional studies - Gene targeting
Functional studies - Insertion mutagenesis
Functional studies - Insertion mutagenesis
How to generate a random insertion mutant collectiongenerate a big population with randomly tagged linesamplify tagged locus with TAIL PCRsequence amplified locusput sequence in a databaseothers interested in the tagged gene/locus can obtain a mutant
Functional studies - Insertion mutants
Transcriptomics and gene expression profiling
Microarrays
Heat map
What you need to know and understand
for the exam and for your life....
... organization of a (eukaryotic) gene
... vector types
... usefuless of genomic sequences
The end