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Roche “454” DNA Sequencing Images adapted from Teri Rambo Mueller Roche Diagnostics Corporation

Roche “454” DNA Sequencing Images adapted from Teri Rambo Mueller Roche Diagnostics Corporation. Adaptor B. Adaptor A. Fluorescent Label. Adaptor allows single DNA molecule to bind bead contained in drop of emulsion. emulsion PCR. Wells are picoliter reaction vessels .

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Roche “454” DNA Sequencing Images adapted from Teri Rambo Mueller Roche Diagnostics Corporation

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  1. Roche “454” DNA Sequencing Images adapted from Teri Rambo Mueller Roche Diagnostics Corporation

  2. Adaptor B Adaptor A Fluorescent Label

  3. Adaptor allows single DNA moleculeto bind bead contained in drop of emulsion. emulsion PCR

  4. Wells are picoliter reaction vessels. Wells have titanium coating. Each well holds 1 bead. Multiple optical fibers fused in array. Light-producing reactions in wells measured by CCD camera.

  5. oxy luciferin + light

  6. Polymerase adds nucleotide Pyrophosphate (PPi) released Sulfurylase: PPi  ATP Luciferase: ATP + luciferin  light

  7. Repeated dNTP flow sequence: T A C G Process continues until defined number of cycles completed.

  8. Bead with clonally amplified DNA A A T C G G C A T G C T A A A A C T G A T T A G C C G T A C G A T T T T C G A C T Primer 4xT homopolymer produces more light Key Sequence

  9. Key Sequence Homopolymer produces more light Repeated dNTP flow sequence: T A C G Process continues until user-defined number of nucleotide flow cycles are completed. A A T C G G C A T G C T A A A A C T G A T T A G C C G T A C G A T T T T C G T A G C A T Anneal Primer

  10. Polymerase adds nucleotide (dATP) • Pyrophosphate is released (PPi) • Sulfurylase creates ATP from PPi • Luciferase hydrolyses ATP and uses luciferin to make light DNA Bead with clonally amplified fragments

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