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 5’ 3’ Dideoxy method of DNA sequencing Annealing * + 35S[dATP] Polymerization and labeling Termination Polyacrylamide/urea gel electrophoresis *- substituded automated DNA sequncing methods Pierce 19.6 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 - 35S instead of O- One of the deoxyriboucleosides triphosphate is labeled with 35S-(α-thio)-triphosphate (or α-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. The sequence information is directly read and electronically stored into the computer, which Pierce 19.8 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 3’ 5’ 5’ 3’ Determination of nucleotide sequence in both chains 1ª fase na análise de dados de sequenciação de 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 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 Hydrophobic regions Hydrophilic regions Hydrophobicity 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.