Biochemistry 412 Overview of Genomics & Proteomics 18 January 2005

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  X174 to human) has doubled approximately every 18 months!

Lander et al (2001) Nature 409, 860.

The Random “Shotgun” DNA Sequencing Strategy >>> Allows sequence information about a target genome to be accumulated rapidly and in a non-biased and semi-automatable fashion.

Random shotgun DNA Sequencing Fragmentation by DNAase I digestion of target DNA in the presence of Mn ++ Anderson (1981) Nucleic Acids Res. 9, 3015.

Random fragmentation yields clones covering the target DNA region (in this case, a portion of the human mitochondrial genome) Anderson (1981) Nucleic Acids Res. 9, Coverage is reasonably complete and uniform Most regions are sequenced more than once, improving overall accuracy

Anderson (1981) Nucleic Acids Res. 9, >>> The recursive, identical steps involved in random shotgun DNA sequencing allowed automation of the sequencing process (even for very large genomes).

Lander et al (2001) Nature 409, 860.

Venter et al (2001) Science 291, 1304.

[Note: individual “B” is Craig Venter!!]

Differences between the Public (Lander et al) and Celera (Venter et al) Human Genome Sequencing Efforts Public Project: Mapped BACs and YACs from the genome first, then shotgun sequenced these (to sort out where the repeats were) Started earlier (~1990) Initial (2001) draft not as accurate as Celera’s (see below)* Finished later (~2003) Final draft more accurate than Celera’s Cost: ca. $3 billion Celera Project: Shotgun sequenced entire human genome in one go Used sequenced end pairs from linking clones to address the repeat problem (also used public project data) Started in the late ‘90s Initial (2001) draft more accurate than public’s (see below)* Quit working before final draft was finished Cost: ca. $300 million**

Differences between the Public (Lander et al) and Celera (Venter et al) Human Genome Sequencing Efforts (footnotes from previous slide) *Celera’s sequence, which was proprietary, incorporated all of the public data, which was available on the internet, so initially Celera’s genome sequence was more complete and accurate than the public consortium’s sequence (Duh!!). At the time, this fact escaped most journalists who reported on the competition with the public consortium’s effort, and the consortium scientists did not help their cause by dumping on Celera’s data! **Applied Biosystems (ABI, Celera’s parent company) more than recouped all of its expenses by selling DNA sequencing machines to -- among others -- the panicked public sequencing consortium members (and also by selling Celera stock to Wall Street). Some observers have even suggested that the entire Celera human genome sequencing effort was nothing more than a Machiavellian marketing ploy by ABI! >>> Nevertheless, the race between the public and private sectors delivered a high quality finished human genome sequence to the scientific community years earlier than would have been the case without such a competition!

Lander et al (2001) Nature 409, 860.