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.

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..” – www.nobel.se

• 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 fidelityError rate of ~ 10-4 to 10-5.

Error rate of ~ 10-9 to 10-12.DNA pol fidelity

Post-transcriptional regulation

Discovering

GENOMICS

TRANSCRIPTOMICS

PROTEOMICS

BIOINFORMATICS

DNA

Pre-mRNA

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

Dideoxynucleoside Sequencing

Dideoxynucleoside Sequencing

Séquençage par des ddNTP Fluorescents

Genome Sequencing Approaches

https://www.roche-applied-science.com/sis/sequencing/flx/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 - Process Overview

454 LifeSciences Sequencer

454 LifeSciences Sequencer

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.

* 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