Transcript Chapter 14 - Genomes and genomics

Chapter 14 Genomes and Genomics
Sequencing DNA
dideoxy (Sanger) method
ddGTP ddATP ddTTP ddCTP
5’TAATGTACG
TAATGTAC
TAATGTA
TAATGT
TAATG
TAAT
TAA
TA
T
Fred Sanger, Nobel prize 1980
Sequencing DNA
dideoxy (Sanger) method
Leroy Hood, Caltech
Fluorescence based sequencing
Norm Dovici – Capillary electrophoresis
Sequencing DNA
dideoxy (Sanger) method
Genomics era: High-throughput DNA sequencing
 The first high-throughput genomics
technology was automated DNA sequencing
in the early 1990.
 TIGR (The Institute for Genomics Research)
1995 – first whole genome sequence, H.
influenza
 Baker’s yeast, Saccharomyces cerevisiae
(15 million bp), was the first eukaryotic
genome to be sequenced.
 In September 1999, Celera Genomics
completed the sequencing of the
Drosophila genome.
Genomics: Completed genomes as 2002
 Currently the genome of over 600 organisms are sequenced:
http://www.genomesonline.org/
This generates large amounts of information to be handled by individual
computers.
Cloning/libraries
BAC, 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
Assembling
contigs
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

century is a century of biotechnology:
Genomics: New sequence information is being
produced at increasing rates. (The
contents of GenBank double every year)
Nucleotides(billion)
21st
8
7
6
5
4
3
2
1
0
1980
1985
1990
Years

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

Glycomics:Global sugar metabolism analysis
1995
2000
How to handle the large amount of information?
Drew Sheneman, New Jersey--The Newark Star Ledger
Answer: bioinformatics and Internet
Bioinformatics history
In1960s:
the birth of bioinformatics
IBM 7090 computer
Margaret
Oakley Dayhoff created:
The first protein database
The 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/)
Homologs
orthologs
paralogs
Question
You 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.
b.
c.
d.
e.
cDNA sequences
Protein sequences
Genomic DNA sequences
Both (a) and (b) will give you equivalent sequence similarity
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
mutant
WT
A “good” microarray
plate
Red = only in treatment
Green = only in normal
Yellow = found in both
Black = found in neither
Results
100’s of genes identified,
those turned on, those turned off
Expression map
red = up regulated
green= down regulated
Question
Microarray technology directly involves:
a.
b.
c.
d.
e.
PCR
DNA sequencing
Hybridization
RFLP detection
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
Bi Molecular
Fluorescence
Complementation
(BMFC)
Citovsky et al., 2006
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