Reference books: Molecular Biology of the Cell, 4th edition, by B. Alberts et al., 2002. Molecular Cell Biology, 5th edition, by H. Lodish et al., 2004.

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Reference books: Molecular Biology of the Cell, 4th edition, by B. Alberts et al., Molecular Cell Biology, 5th edition, by H. Lodish et al., Molecular Biology of the gene, 5th edition, J. Watson et al, 2004 Planches à voir Tazi 1. PPT METHODOLOGIE.PDF

Watson & Crick – “…the secret of life” Watson: a zoologist, Crick: a physicist “In 1947 Crick knew no biology and practically no organic chemistry or crystallography..” – Applying Chagraff’s rules and the X- ray image from Rosalind Franklin, they constructed a “tinkertoy” model showing the double helix Their 1953 Nature paper: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick with DNA model Rosalind Franklin with X-ray image of DNA

DNA RNA Protein Replication Transcription Translation RNA pol fidelity Error rate of ~ to Error rate of ~ to DNA pol fidelity Post-transcriptional regulation

Discovering GENOMICS TRANSCRIPTOMICS PROTEOMICS BIOINFORMATICS

DNA Pre-mRNA mRNA Proteins Metabolites (Genome) (Transcriptome) (Proteome) (Metabolome)

Steps in DNA Sequencing and Genotyping Selecting part of genome to sequence or genotype Amplifying nucleic acid Determine sequence of one or a succession of nucleic acids: –Chemical reaction format –Read out format Resequencing vs de novo sequencing

Sequence Tagget sites (STSs) Short segments of unique DNA sequence along every chromosome

Dideoxynucleoside Sequencing

Séquençage par des ddNTP Fluorescents

Genome Sequencing Approaches

Multimedia presentation

Pyrosequencing Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

Pyrosequencing - Solid Phase Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

Pyrosequencing - Liquid Phase Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

Pyrogram Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

454 LifeSciences Sequencer

454 LifeSciences Sequencer - Process Overview

454 LifeSciences Sequencer

Applications of Ultra-Low-Cost Sequencing Sequencing individual human genomes as component of preventive care. Genotype-phenotype associations Comprehensive gene expression profiling in vitro and in situ at all stages of development of a multicellular organism Comprehensive analysis of mutations present in cancer clones. Mitochondrial heteroplasmy Microbial diversity (metagenomic studies) Shendure N. Advanced sequencing technology: methods and goals. Nat Rev Gen 2004

3 billion bases 20,000 – 30,000 genes 3000 nucleotide/gene (on average) 99,9% nucleotide similarity to each other 99% nucleotide similarity to chimpanzees Less than 2 % codes for proteins Chromosome 1 has the most genes Chromosome Y has the fewest genes

Variations in human genome Single Nucleotide polymorphisms Microsatelitte sequences and variation "Microsatellites" are defined as loci (or regions within DNA sequences) where short sequences of DNA are repeated in tandem arrays. This means that the sequences are repeated one right after the other. The lengths of sequences used most often are di-, tri-, or tetra-nucleotides.DNA

* Protein Coding sequences represent less than 2% of human genome * More than 50% of human genome sequences are repetitive sequences

CpG islands density on each chromosome