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Biochemistry 412 Overview of Genomics & Proteomics 20 January 2004. DNA Sequencing & the Human Genome Project. Timeline: The Foundations of Genomics 1953 • Model for 3D structure of DNA - J. Watson & F. Crick • First protein sequence (insulin) - F. Sanger
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Biochemistry 412 Overview of Genomics & Proteomics 20 January 2004
DNA Sequencing & the Human Genome Project
Timeline: The Foundations of Genomics 1953 • Model for 3D structure of DNA - J. Watson & F. Crick • First protein sequence (insulin) - F. Sanger 1965 • First RNA sequences - R W. Holley & colleagues; F. Sanger & colleagues 1970 • Restriction endonucleases discovered - D. Nathans & H. O. Smith 1972 • First recombinant DNA molecule - P. Berg & colleagues 1975 • “Plus-minus” method of DNA sequencing - F. Sanger & A. R. Coulson 1977 • Chemical method of DNA sequencing - A. Maxam & W. Gilbert • Dideoxy method of DNA sequencing - F. Sanger & A. R. Coulson • First bioinformatics software for DNA sequences - R. Staden 1978 • Single-stranded phage vectors developed - J. Messing & colleagues 1980 • “Shotgun cloning” strategy for DNA sequencing - J. Messing & colleagues; F. Sanger & colleagues 1981 • Random shotgun cloning method developed - S. Anderson 1985 • Polymerase chain reaction (PCR) method developed - K. Mullis 1986 • Development of first automated DNA sequencer - L. Hood & colleagues >>> For the past 25+ years, the size of the largest genome sequenced (from PhiX174 to human) has doubled approximately every 18 months!
The human genome sequence is finished…. >>> But what other genome-based studies have been enabled by this achievement? Some examples: • Human variation and evolution (e. g., “SNPs”) • Somatic mutations (e. g., loss-of-heterozygosity in cancer) • RNA expression profiling (cf. “DNA chips”) • Methylation patterns (e. g., epigenetics and gene silencing)
Single Nucleotide Polymorphisms (“SNPs”)
Microarrays (DNA Chips)
Note: 4N masks required to make an array of oligonucleotides each of length N. Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A.91, 5022.
Note: this is the photolabile blocking group, “X”, indicated schematically in Figure 1. Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A.91, 5022.
Key feature: known oligo sequence at each “address” on the chip. Lipshutz et al (1999) Nature Genet. (suppl.)21, 20.
Note: caloric restriction gene chip experiment w/ rats. Ref: Lee et al (1999) Science285, 1390.
“Proteomics” The study of the complete complement of proteins found in an organism
“Degrees of Freedom” for Protein Variability Covalent Modifications in Proteins • Post-translational modifications (e.g., phosphorylation, glycosylation, etc.) - more than 200 such modifications are known, and they can occur at multiple sites in a single protein • Alternative splicing of a primary transcript - in extreme cases, a single gene can produce tens of thousands of different mRNAs! • Proteolytic processing • Protein aging Thus, there are probably many millions of different proteins in our bodies!!
Other realities of proteins • They have “personalities”: each behaves differently. • They exist in different concentrations, ranging over a million-fold. • It will be extremely difficult to even identify them all (see previous slide). Take-home message: Proteomics presents challenges that are orders-of-magnitude more difficult than those presented by genomics!
“Classic” Proteomics: 2-Dimensional Gel Electrophoresis <-------- separation by charge --------> <--------- separation by size ----------->
Protein chips Pandey & Mann (2000) Nature405, 837.
Yeast Two-Hybrid System (Song and Fields) Pandey & Mann (2000) Nature405, 837.