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!

Lander et al (2001) Nature 409, 860.

Venter et al (2001) Science 291, 1304.

Lander et al (2001) Nature 409, 860.

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”)

Roses (2000) Nature 405, 857.

Microarrays (DNA Chips)

Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 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, Note: this is the photolabile blocking group, “X”, indicated schematically in Figure 1.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20. Key feature: known oligo sequence at each “address” on the chip.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.

RNA Profiling

Lockhart & Winzeler (2000) Nature 405, 827.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.

Lockhart & Winzeler (2000) Nature 405, 827.

Bassett et al (1999) Nature Genet. (suppl.) 21, 51.

Ref: Lee et al (1999) Science 285, Note: caloric restriction gene chip experiment w/ rats.

Lee et al (1999) Science 285, 1390.

Kapranov et al (2002) Science 296, 916.

“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

Pandey & Mann (2000) Nature 405, 837.

Protein chips

Pandey & Mann (2000) Nature 405, 837. Yeast Two-Hybrid System (Song and Fields)