DNA sequencing - ULisboabmg.fc.ul.pt/Disciplinas/FundBiolMolec/10DNA sequencing.pdf · The dideoxy...
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Transcript of DNA sequencing - ULisboabmg.fc.ul.pt/Disciplinas/FundBiolMolec/10DNA sequencing.pdf · The dideoxy...
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
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
Dideoxy method of DNA sequencing
Pierce 19.6
Annealing
Polymerization and labeling
Termination
Polyacrylamide/urea gel electrophoresis
+ 35S[dATP]*
*- substituded automatedDNA 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
35S35S--(a(a--thio)thio)--deoxynucleosidedeoxynucleoside triphosphatetriphosphate
One of the deoxyriboucleosides triphosphate is labeled with 35S-(α-thio)-triphosphate(or α-32P-triphosphate), allowing neo-synthesized chain to be detected
- 35S instead of O-
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
Printout from an automatic sequencer thatuses fluorescent dyes
Suzuki 12-24
N, represents a base that cannot be assigned
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)
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 potential genes
Hydrophobicity profileH
ydro
phob
icity
Aminoacids residues
Hydrophobic regions
Hydrophilic regions
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