dna sequencing - ulisboabmg.fc.ul.pt/disciplinas/gbm/aulas/5dnasequencing.pdf · dna sequencing...
TRANSCRIPT
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Dideoxy-terminating sequencingor
Sanger “dideoxy sequencing”
DNA sequencing
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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)
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The dideoxy sequencingrequires a
special substratefor DNA synthesis
dNTP vs ddNTP
The dideoxy method of DNA sequencing isbased on the termination of DNA synthesis
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Nucleotides analogues(DNA chain terminators)
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DNA template:cloned DNA or PCR product
Vectors contain universal sequencing primer sites on either sideof the site where DNA will be inserted
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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
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5’
3’
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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
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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 -
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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
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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
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Each well is a single sample
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Printout from an automatic sequencer thatuses fluorescent dyes
Suzuki 12-24
N, represents a base that cannot be assigned
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DNA sequence data on manual and automaticsequencing
5’ 3’
5’
3’
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Determination of nucleotide sequence in both chains
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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)
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6-phase ORF Map(DNA Strider)
4 kb DNA fragment
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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
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Restriction map
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Specific sequences and signs
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3-phase Translation
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1-phase-Translation
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Protein sequence
645 aa
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Aminoacid usage
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Hydrophobicity profileH
ydro
phob
icity
Aminoacids residues
11 residues
Hydrophobic regions
Hydrophilic regions
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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