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