Chapter 14 Genomes and Genomics

Sequencing DNAdideoxy (Sanger) method

ddGTP ddATP ddTTP ddCTP

5’TAATGTACG TAATGTACTAATGTATAATGTTAATGTAATTAATAT

Fred Sanger, Nobel prize 1980

Sequencing DNAdideoxy (Sanger) method

Leroy Hood, CaltechFluorescence based sequencing

Norm Dovici – Capillary electrophoresis

Sequencing DNAdideoxy (Sanger) method

Genomics era: High-throughput DNA sequencing

The first high-throughput genomics technology was automated DNA sequencing in the early 1990.

In September 1999, Celera Genomics completed the sequencing of the Drosophila genome.

Baker’s yeast, Saccharomyces cerevisiae (15 million bp), was the first eukaryotic genome to be sequenced.

TIGR (The Institute for Genomics Research) 1995 – first whole genome sequence, H. influenza

Genomics: Completed genomes as 2002

Currently the genome of over 600 organisms are sequenced:

This generates large amounts of information to be handled by individual computers.

http://www.genomesonline.org/

Cloning/librariesBAC, YAC and ESTs

• BAC = bacterial artificial chromosome– 150 kb, replicate in E.coli

• YAC = yeast artificial chromosome– 150 kb -1.5 Mb, replicate in yeast

Assemblingcontigs

Ordered-clone Sequencing

Clones ordered by restriction enzyme sites

Annotation

• ORF – open reading frame

• EST- Expressed sequence tag – Based on mRNA

• Comparative genomics

The trend of data growth

01

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1980 1985 1990 1995 2000

Years

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21st century is a century of biotechnology:

Microarray: Global expression analysis: RNA levels of every gene in the genome analyzed in parallel.

Proteomics:Global protein analysis generates by large mass spectra libraries.

Metabolomics:Global metabolite analysis: 25,000 secondary metabolites characterized

Genomics: New sequence information is being produced at increasing rates. (The

contents of GenBank double every year)

Glycomics:Global sugar metabolism analysis

How to handle the large amount of information?

Drew Sheneman, New Jersey--The Newark Star Ledger

Answer: bioinformatics and Internet

Bioinformatics history

IBM 7090 computer

In1960s: the birth of bioinformatics

Margaret Oakley Dayhoff created:The first protein databaseThe first program for sequence assembly

There is a need for computers and algorithms that allow: Access, processing, storing, sharing, retrieving, visualizing, annotating…

DNA (nucleotide sequences) databases

They are big databases and searching either one should produce similar results because they exchange information routinely.

-GenBank (NCBI): www.ncbi.nlm.nih.gov

-Arabidopsis: (TAIR) www.arabidopsis.org

Specialized databases:Tissues, species… -ESTs (Expressed Sequence Tags)

~at NCBI ~at TIGR

- ...many more!

Comparative genomics

BLAST – basic local alignment and search tool(http://www.ncbi.nlm.nih.gov/)

Homologsorthologsparalogs

QuestionYou are a researcher who has tentatively identified a human homolog of a yeast gene. You determine the DNA sequence of cDNAs of both your yeast gene and the human gene and decide to compare the gene sequences, as well as the predicted protein sequence of each, using alignment software. You would expect the greatest sequence identity from comparisons of the:

a. cDNA sequencesb. Protein sequencesc. Genomic DNA sequencesd. Both (a) and (b) will give you equivalent sequence similaritye. All will give equivalent sequence similarity

What is a microarray?

Types of Arrays

• Expression Arrays – cDNA– Genome

• Affymetrix (GeneChip®)

• Agilent

• Tiling arrays

Overview of Microarrays

Transcription Profiling of a mutant

WT

mutant

A “good” microarray plate

Red = only in treatment

Green = only in normal

Yellow = found in both

Black = found in neither

ResultsResults

100’s of genes identified,

those turned on, those turned off

Expression mapred = up regulatedgreen= down regulated

Question

Microarray technology directly involves:

a. PCR

b. DNA sequencing

c. Hybridization

d. RFLP detection

e. None of the above

Protein – protein interactions

• ChIP (chomatin immunoprecipitation)

• Yeast two hybrid

• Bi Molecular Fluorescence Complementation (BMFC)

ChIP and ChIP- chip

Yeast two hybrid

Citovsky et al., 2006

Bi Molecular Fluorescence Complementation(BMFC)

Reverse genetics

• Gene knockouts

• RNAi

• Overexpression

• Altered expression

Summary

• DNA Sequencing and the rise of genomics

• Annotation of genome sequence– Comparative genomics– Functional genomics

• Protein-protein interactions

• ESTs

• Reverse genetics