DNA sequencing. Dideoxy-terminating sequencing or Sanger dideoxy sequencing

Transcription

1 DNA sequencing Dideoxy-terminating sequencing or Sanger dideoxy sequencing

2 Tools DNA template (single stranded) Specific primer (usually 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)

3 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

4 Nucleotides analogues (DNA chain terminators)

5 DNA template: cloned DNA or PCR product Vectors contain universal sequencing primer sites on either side of the site where DNA will be inserted

6 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

7 5 3

8 Dideoxy method of DNA sequencing + 35 S[dATP] * Annealing Polymerization and labeling Termination Polyacrylamide/urea gel electrophoresis Pierce 19.6 *- substituded automated DNA sequncing methods

9 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

10 35S-(a (a-thio) thio)-deoxynucleoside triphosphate - 35 S instead of O - One of the deoxyriboucleosides triphosphate is labeled with 35 S-(α-thio)-triphosphate (or α- 32 P-triphosphate), allowing neo-synthesized chain to be detected

11 Each of the four ddntps is tagged with a fluorescent dye The dideoxy sequencing method can be automated 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. Pierce 19.8 The sequence information is directly read and electronically stored into the computer, which converts it into the complementary- target- sequence

12

13 Each well is a single sample

14 Printout from an automatic sequencer that uses fluorescent dyes N, represents a base that cannot be assigned Suzuki 12-24

15 DNA sequence data on manual and automatic sequencing

16 Determination of nucleotide sequence in both chains

17 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)

18 6-phase ORF Map (DNA Strider) 4 kb DNA fragment

19 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

20 Restriction map

21 Specific sequences and signs

22 3-phase Translation

23 1-phase-Translation

24 Protein sequence 645 aa

25 Aminoacid usage

26 Hydrophobicity profile Hydrophobicity Hydrophobic regions Hydrophilic regions Aminoacids residues 11 residues

27 Sequence Analysis Databases Major Sequence Repositories GenBank or NCBI (all known nucleotide and protein sequences) Ensembl (all known nucleotide and protein sequences) TIGR Gene Indices (non-redundant, gene oriented clusters) 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) Genetic Maps GBD (Human genes and genomic maps) Genomic Databases Flybase (Drosophyla sequences and genomic information) MGD (Mouse genetics and genomics)