Pyrosequencing MCQ

Q70.Which of the following sentences is not true for pyrosequencing? (1) It is based on detection of pulses of chemiluminescence (2) Requires the activity of enzyme sulfurylase (3) Requires addition of dideoxynucleotides (4) None of these

Q70.Which of the following sentences is not true for pyrosequencing?

(1) It is based on detection of pulses of chemiluminescence
(2) Requires the activity of enzyme sulfurylase
(3) Requires addition of dideoxynucleotides
(4) None of these

Pyrosequencing False Statement Revealed

Pyrosequencing detects DNA incorporation via chemiluminescence from pyrophosphate release, but it does not use dideoxynucleotides like Sanger sequencing.

Correct Answer

Option (3) Requires addition of dideoxynucleotides is not true for pyrosequencing.

Pyrosequencing is a sequencing-by-synthesis method using regular deoxynucleotides (dNTPs), not chain-terminating ddNTPs.

Option Breakdown

Option Statement Explanation
(1) Detection of chemiluminescence pulses True Light emitted via luciferase converts ATP (from PPi via sulfurylase) into detectable flashes proportional to incorporated bases.
(2) Requires sulfurylase activity True ATP sulfurylase converts released PPi + APS to ATP, enabling the luciferin-luciferase light reaction.
(3) Requires dideoxynucleotides False Uses only dNTPs added sequentially; ddNTPs (lacking 3′-OH) are Sanger-specific for termination, not pyrosequencing.
(4) None of these False Option 3 is untrue, so this doesn’t apply.

Pyrosequencing Mechanism

Single-stranded DNA template hybridizes with primer; DNA polymerase, sulfurylase, luciferase, apyrase, and substrates wait in reaction mix.

dNTPs added one type at a time: incorporation releases PPi → ATP → oxyluciferin + light (peak height shows homopolymer length); apyrase degrades unused dNTPs/PPi.

Pyrograms display sequence as light peak patterns, ideal for short reads, methylation detection, and SNP genotyping.

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