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Genomic DNA Cloning

Genomic DNA Cloning. Plasmid DNA Ligation. DNA Cloning. Shotgun sequencing di un intero genoma. Frammentazione DNA genomico in piccoli pezzi (ca.1000 bp) Clonaggio in plasmidi Sequenza dell’ inserto dalle due estremità Allineamento computerizzato delle sequenze Quanto sequenziare?

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Genomic DNA Cloning

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  1. Genomic DNA Cloning

  2. Plasmid DNA Ligation

  3. DNA Cloning

  4. Shotgun sequencing di un intero genoma • Frammentazione DNA genomico in piccoli pezzi (ca.1000 bp) • Clonaggio in plasmidi • Sequenza dell’ inserto dalle due estremità • Allineamento computerizzato delle sequenze • Quanto sequenziare? • 5-7genomi equivalenti

  5. Sequenziare un largo numero di inserti plasmidici (5-10 genomi equivalenti) Allineamento computerizzato delle sequenze in contiguous assemblies (contigs): 1 2 3 Shotgun sequencing chiudere i gaps e ordinare in un solo contig (1-2-3, 1-3-2, 2-1-3?) Isolamento cloni di chiusura, PCR.

  6. Open reading frame (ORF) Stop Start 5’ …C T C A A T G G G T A C G T A G G AT C G G G A A T C G T A C A G G A A C G T T T G A A A T C G... 3’ … G A G T T A C C C A T G C A T C C T A G C CC T T A G C A T G T C C T T G C A A A C T T T A G C... • Identificazione geni (ORFs,Open Reading Frames) • Criteri: lunghezza, codon usage, • Sequenze Shine-Dalgarno

  7. Dimensioni genomi • Batteri: Mycoplasma genitalium 0.58 milioni bp (Mb), 467 geni Escherichia coli 4.64 Mb, 4289 geni • Eucarioti • S. cerevisiae:12 Mb, 6241 geni • Drosophila melanogaster: 180 Mb; 13,601 geni • Homo sapiens: ~3200 Mb; ~30,000 geni

  8. Dimensioni e numero dei geni • Average gene size: • Bacteria: 1100 bp • Yeast: ~1200 bp • Worm: ~5000 bp • Human: ~27,000 bp (range up to 2.4 Mb) • Distance between genes: • Bacteria: 118 bp • Yeast: ~700 bp • Human: range from overlapping to ~1 Mb

  9. Sanger Sequencing

  10. 1. A single strand of DNA to be sequenced (yellow) is hybridized to a 5’ end labeled synthetic deoxynucleotide primer(Brown). • 2. The primer is elongated using DNA polymerase in four • separate reaction mixtures containing four normal • deoxynucleotide triphosphates (dNTPs) plus one of • dideoxynucleotide triphosphate (ddNTPs) in a ratio of • 100 :1.

  11. 2: Large-Scale Pyrosequencing: sequencing at the speed of light

  12. 2: Large-Scale Pyrosequencing: • Gel free • Nucleotides are label free • Parallelism

  13. Pyrosequencing summary • •Emulsion PCR. Eliminates library construction and DNA prep • •Drop one bead/well into a “picotiter” plate (1.6 million wells) • •Image reactions in a flow cell • with CCD camera

  14. A primer is hybridized to a single stranded, PCR amplified, DNA template and incubated with: •DNA polymerase •ATP sulfurylase •luciferase •apyrase •adenosine 5´ phosphosulfate (APS) •luciferin

  15. Each DNA fragment is amplified and attached to a bead separately (one bead/fragment). Each bead is added to a fibre-optic well.

  16. The first dNTP is added to the reaction. And is incorporated or not incorporation is accompanied by release of pyrophosphate (PPi)

  17. ATP sulfurylase converts PPi to ATP, which drives the luciferase-mediated conversion of luciferin to oxyluciferin This generates light, detected by a charge coupled device (CCD) camera and seen as a peak. The light signal is proportional to the number of nucleotides incorporated.

  18. Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

  19. Addition of dNTPs: one at a time. The nucleotide sequence is determined from the signal peak in the pyrogram

  20. 2: Large-Scale Pyrosequencing A computer can read the light pattern from billions of wells simultaneously. (Sequencing of a bacterial genome in 7h).

  21. Bioinformatics and medicine Your chip analysis suggests stress

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