Einführung in die Genetik

Prof. Dr. Kay Schneitz (EBio Pflanzen)http://plantdev.bio.wzw.tum.deschneitz@wzw.tum.de

Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/sysbiolclaus.schwechheimer@wzw.tum.de

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