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#pyrosequencing

2 public questions tagged with this topic.

Sequencing

What does luciferase do in pyrosequencing?

In pyrosequencing, light emission provides quantitative readout of nucleotide incorporation. Once ATP is generated from released pyrophosphate by ATP sulfurylase, firefly luciferase catalyzes the ATP-dependent oxidation of D-luciferin to oxyluciferin, producing a flash of yellow-green light. Emission is captured by a sensitive CCD camera, and peak height correlates with number of identical bases added consecutively. DNA polymerase extends strand, ATP sulfurylase synthesizes ATP, apyrase degrades unincorporated substrates. The distinct role of luciferase is generating photons that enable real-time identification of incorporated nucleotides.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Sequencing

In pyrosequencing, PPi is converted to ATP by:

During pyrosequencing, the enzymatic cascade links DNA synthesis to bioluminescence. After polymerase releases pyrophosphate, ATP sulfurylase catalyzes reaction between PPi and adenosine 5' phosphosulfate, APS, forming ATP. This newly generated ATP serves as substrate for luciferase in the next step, distinct from background ATP. Luciferase itself does not produce ATP; it consumes ATP to generate light, while apyrase removes residual nucleotides and ATP between cycles. DNA polymerase only incorporates dNTPs. Therefore conversion of PPi to ATP is specifically mediated by ATP sulfurylase bridging synthesis and luminescence.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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