Biochemistry 412

Overview of Genomics & Proteomics

18 January 2005

Biochemistry 694:412Proteomics and Functional Genomics

Spring 2005CABM 208

9:50 – 11:10 AM Tu/Fri

Syllabus of Lectures

Date Topic Lecturer

T, Jan. 18th Course introduction and overview of genomics & proteomics GTM + SAF, Jan. 21st Genomics and proteomics (con’d) SA

T, Jan. 25th Tools of bioinformatics: Blast, PsiBlast, DNA sequence databases GTMF, Jan. 28th Comparative biology based on genomic sequence analysis GTM

T, Feb. 1st Analytical and preparative protein chemistry I SAF, Feb. 4th Analytical and preparative protein chemistry II SA

T, Feb. 8th Protein sequence and structure databases GTMF, Feb. 11th Exam I

T, Feb. 15th Protein folding SAF, Feb. 18th Conformational properties of proteins SA

T, Feb. 22nd Protein mass spectrometry SAF, Feb. 25th Protein NMR spectroscopy I GTM

T, Mar. 1st Protein NMR spectroscopy II GTMF, Mar. 4th Biophysics of amide HD exchange GTM

T, Mar. 8th Protein X-ray crystallography GTMF, Mar. 11th Exam II

T, Mar. 15th Spring BreakF, Mar. 18th Spring Break

Biochemistry 694:412Proteomics and Functional Genomics

Spring 2005CABM 208

9:50 – 11:10 AM Tu/Fri

Syllabus of Lectures (con’d)

Date Topic Lecturer

T, Mar. 22nd Enzyme kinetics I GTMF, Mar. 25th Enzyme kinetics II GTM

T, Mar. 29th Protein-protein interactions I SAF, Apr. 1st Protein-protein interactions II SA

T, Apr. 5th DNA microarrays SAF, Apr. 8th RNA interference SA

T, Apr. 12th Structural Genomics I GTMF, Apr. 15th Structural Genomics II GTM

T, Apr. 19th Exam IIIF, Apr. 22nd Student Presentations

T, Apr. 26th Student PresentationsF, Apr. 29th Student Presentations

The Student Presentations and Exams I - III will each count for 25% of your grade. Therewill be no final exam.

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 andsemi-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, 3015.

• Coverage is reasonably complete and uniform• Most regions are sequenced more than once, improving overall accuracy

Anderson (1981) Nucleic Acids Res. 9, 3015.

>>> 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.

Lander et al (2001) Nature 409, 860.

Lander et al (2001) Nature 409, 860.

Lander et al (2001) Nature 409, 860.

Venter et al (2001) Science 291, 1304.

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.