DNA sequencing

Dideoxy-terminating sequencing
or
Sanger dideoxy sequencing

Tools

DNA template (single stranded)

Specific primer (usually 17-23 mer, free 3-OH)
dNTPs
DNA polymerase
capacity of polymerizing ddNTPs and other
nucleotides analogues
proofreading activity (facultative)
variable processivity (ex 300 nts/min)
Ex. T7 DNA polymerase, Sequenase

ddNTPs (dideoxyribonucleotides)

The dideoxy method of DNA sequencing is

based on the termination of DNA synthesis

The dideoxy sequencing

requires a
special substrate
for DNA synthesis
dNTP vs ddNTP

Nucleotides analogues
(DNA chain terminators)

DNA template:
cloned DNA or PCR product

Vectors contain universal sequencing primer sites on either side

of the site where DNA will be inserted

Single-stranded templates
for DNA sequencing can be provided

Ex: M13 filamentous phage

A universal sequencing primer can be used to sequence many different template DNAs

Dideoxy method of DNA sequencing

Annealing
+ 35S[dATP]

*
Polymerization and labeling

Termination

Polyacrylamide/urea gel
electrophoresis

*- substituded automated
Pierce 19.6

DNA sequncing methods

Labelling of the neo-synthesized chain:

Manual DNA sequencing
Radioactive labeling of a deoxynucleoside
triphosphate

Automated DNA squencing

Fluorescence labeling of ddNTP or primer with
different fluorophores

35S-(a-thio)-deoxynucleoside triphosphate

- 35S instead of O-

One of the deoxyriboucleosides triphosphate is labeled with 35S-(-thio)-triphosphate

(or -32P-triphosphate), allowing neo-synthesized chain to be detected

The dideoxy sequencing

method can be automated
Each of the four ddNTPs is
tagged with a fluorescent dye

Fluorescent dye detected by using

a laser beam and a detector

Denaturated DNA products are

mixed and laoded into a single
well on an electrophoresis gel.

Pierce 19.8

The sequence information is directly read and

electronically stored into the computer, which
converts it into the complementary- target- sequence

Each well is a single sample

Printout from an automatic sequencer that

uses fluorescent dyes

N, represents a base that cannot be assigned

Suzuki 12-24

DNA sequence data on manual and automatic

sequencing

Determination of nucleotide sequence in both chains

1 fase na anlise de dados de sequenciao

de DNA

Armazenamento de dados
Sobreposio e alinhamento de sequncias nucleotdicas de
diferentes clones ou origens
Comparao de sequncias nucleotdicas e peptdicas num banco
de dados
Anlise de dados:

Sequncia da cadeia complementar

Carta de restrio
ORFs
Sequncia de peptdica
Perfis de hidrofobicidade
Pesquisa de sequncias especficas
Pesquisa de regies com potencial para a formao de estruturas
secundrias: stem-loops, palindromas etc
% G/C
codon usage (codon preference)

6-phase ORF Map

(DNA Strider)

4 kb DNA fragment

Any piece of DNA has six possible reading

frames, three in each direction

Computer has scanned a 9 kb DNA fragment looking for ORFs (potential genes)
Two ORFs, 1 and 2, are the most likely candidates as potential genes

Suzuki 12-24

Restriction map

Specific sequences and signs

3-phase Translation

1-phase-Translation

Protein sequence

645 aa

Aminoacid usage

Hydrophobicity profile

Hydrophobicity

Hydrophobic regions

Hydrophilic regions

Aminoacids residues

11 residues

Sequence Analysis Databases

Major Sequence Repositories

GenBank or NCBI (all known nucleotide and protein sequences)
www.ncbi.nlm.nih.gov/Web/Genbank/
Ensembl (all known nucleotide and protein sequences)
www.ensembl.org/index.html

TIGR Gene Indices (non-redundant, gene oriented clusters)

www.tigr.org/tdb/tdb.html

Gene Expression
BodyMap (Human and mouse gene expression data)
bodymap.ims.u-tokyo.ac.jp

Gene Identification and Structure

EID (Protein-coding, intron-containing genes)
mcb.harvard.edu/gilbert/EID/
Exint (Exon-intron structure of eukaryotic genes)
intron.bic.nus.edu.sg/exint/extint.html
TRRD (Regulatory regions of eukaryotic genes)
www.mgs.bionet.nsc.re/mgs/dbases/trrd4/

Genetic Maps
GBD (Human genes and genomic maps)
www.gbd.org

Genomic Databases
Flybase (Drosophyla sequences and genomic information)
www.fruitfly.org
MGD (Mouse genetics and genomics)
www.informatics.jax.org