Post on 02-Dec-2018
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