http://www.ncbi.nlm.nih.

gov/

Bioinformatics and Gene Hunting Strategies

Library Construction
Cut into pieces
Bioinformatics and Gene Hunting Strategies
Insertion
into vectors
(Genomic library)
Protein purification and microsequencing
Insertion
cDNAs
into vectors
(cDNA library)

cDNA library
Genomic library

clone

Bioinformatics and Gene Hunting Strategies

Protein purification and microsequencing

cDNA library
Genomic library
library

1
 Protein purification and microsequencing Protein purification and microsequencing

Enzyme
activity Protein microsequencing
by Edmen degradation

Enzyme
activity

Protein purification and microsequencing Protein purification and microsequencing

Protein microsequencing
by Edmen degradation

Degenerate DNA sequence => isotope-labelled DNA probe

DNA hybridization

cDNA library
cDNA clones

Degenerate DNA sequence => isotope-labelled DNA probe

DNA hybridization DNA hybridization

cDNA library

+
probe
?

DNA
sequencing

2
 Frederick Sanger

chain termination method

Bioinformatics and Gene Hunting Strategies Bioinformatics and Gene Hunting Strategies

Protein purification and microsequencing Protein purification and microsequencing

hybridization hybridization
Sequence database

cDNA library cDNA library

Genomic library Genomic library

Bioinformatics and Gene Hunting Strategies

Protein purification and microsequencing

In silico hybridization
(Database search)

Sequence database

cDNA library
Genomic library

3
 BLAST (Basic Local Alignment Search Tool)
Bioinformatics and Gene Hunting Strategies

probe
Gene Myers
Protein purification and microsequencing
In silico hybridization
(Database search) Gene Myers
BLAST

Sequence database
database

BLAST (Basic Local Alignment Search Tool)

BLAST

BLAST BLAST

4
 BLAST (Basic Local Alignment Search Tool)

Database:
Protein
EST
Genome

Database: protein database

Database:
Protein
EST
Gene cloning Complete
Genome DNA
sequencing

5
Database: protein database

Database:
Protein
EST
Gene cloning Complete
DNA Genome
sequencing

Protein
sequence

Database: EST database Database: EST database

(expression sequence tag)

J. Craig Venter
cDNA library Partial
c-DNA
sequencing
for all clones

Protein
sequence

6
DNA sequencer

Single gene clone m-RNA database

Protein database

cDNA library

cDNA library

EST database

m-RNA => cDNA

Random Priming

7
 cDNA library

EST and cDNA microarray

Protein purification and microsequencing Protein purification and microsequencing

Enzyme
activity Protein microsequencing
by Edmen degradation

Enzyme
activity

8
Protein purification and microsequencing m-RNA => cDNA library (EST database)

Protein microsequencing
by Edmen degradation

Protein internal partial sequencing Protein internal partial sequencing

Protein internal partial sequencing m-RNA => cDNA library (EST database)

9
 Single gene clone m-RNA database
Protein database
Database:
Protein
EST
Genome cDNA library

EST database

Yeast Artificial Chromosomes

Genome library

Genome database

Yeast Artificial Chromosomes Bacterial Artificial Chromosomes

10
Clone contig Database: Genome database

???

Database: Genome database Mapping Genome

'top-down' or 'clone-by-clone' approach 'top-down' or 'clone-by-clone' approach

???

11
Database: EST database Database: Genome database
(expression sequence tag)
J. Craig Venter
J. Craig Venter

"I expect that between 50 and 100 genomes will have been
completed worldwide by the end of the decade," says J. Craig Venter

Clone contig 'bottom-up' approach: whole genome shotgun

???

Database: Genome database http://www.ncbi.nlm.nih.gov/genomes/static/EG_T.html

J. Craig Venter

"I expect that between 50 and 100 genomes will have been
completed worldwide by the end of the decade," says J. Craig Venter

12
Integrated database Integrated database

Integrated database http://www.ncbi.nlm.nih.gov/

13