Dideoxy-terminating sequencingor

Sanger “dideoxy sequencing”

DNA 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 sequencingrequires a

special substratefor DNA synthesis

dNTP vs ddNTP

The dideoxy method of DNA sequencing isbased on the termination of DNA synthesis

Nucleotides analogues(DNA chain terminators)

DNA template:cloned DNA or PCR product

Vectors contain universal sequencing primer sites on either sideof the site where DNA will be inserted

Single-stranded templatesfor DNA sequencing can be provided

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

Ex: M13 filamentous phage

5’

3’

Dideoxy method of DNA sequencing

Pierce 19.6

Annealing

Polymerization and labeling

Termination

Polyacrylamide/urea gel electrophoresis

+ 35S[dATP]*

*- substituded by automatedDNA sequencing methods with fluorophores

35S35S--(a(a--thio)thio) --deoxynucleosidedeoxynucleoside triphosphatetriphosphate

One of the deoxyriboucleosides triphosphate is labeled wit h 35S-(αααα-thio)-triphosphate(or αααα-32P-triphosphate), allowing neo-synthesized chain to be d etected

- 35S instead of O -

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

The dideoxy sequencingmethod can be automated

Pierce 19.8

Each of the four ddNTPs istagged with a fluorescent dye

Denaturated DNA products aremixed and laoded into a single well on an electrophoresis gel.

The sequence information is directly read andelectronically stored into the computer, whichconverts it into the complementary- target- sequence

Fluorescent dye detected by usinga laser beam and a detector

Each well is a single sample

Printout from an automatic sequencer thatuses fluorescent dyes

Suzuki 12-24

N, represents a base that cannot be assigned

DNA sequence data on manual and automaticsequencing

5’ 3’

5’

3’

Determination of nucleotide sequence in both chains

1ª fase na análise de dados de sequenciaçãode DNA

• Armazenamento de dados• Sobreposição e alinhamento de sequências nucleotídicas de

diferentes clones ou origens• Comparação de sequências nucleotídicas e peptídicas num banco

de dados• Análise de dados:

– Sequência da cadeia complementar– Carta de restrição– ORFs– Sequência de peptídica– Perfis de hidrofobicidade– Pesquisa de sequências específicas– Pesquisa de regiões com potencial para a formação de estruturas

secundárias: 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 readingframes, three in each direction

Suzuki 12-24

Computer has scanned a 9 kb DNA fragment looking for ORFs (potential genes)

Two ORFs, 1 and 2, are the most likely candidates as pot ential genes

Restriction map

Specific sequences and signs

3-phase Translation

1-phase-Translation

Protein sequence

645 aa

Aminoacid usage

Hydrophobicity profileH

ydro

phob

icity

Aminoacids residues

11 residues

Hydrophobic regions

Hydrophilic regions

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