A Level Biology: Pyrosequencing
How many types of activated nucleotides are used in a pyrosequencing reaction?
1
What are the 2 substrates used in pyrosequencing?
APS and luciferin
Give the 4 activated nucleotides used one group at a time in pyrosequencing:
ATP, TTP, CTP, GTP
What are the 4 enzymes used in pyrosequencing?
DNA polymerase, ATP sulfurylase, luciferase, apyrase
Explain how the pyrosequencing reaction produces photons of light:
a compilatory base pair is added to the single strand of DNA using an activated nucleotide and DNA polymerase which produces PPi as a result. PPi is converted to ATP by ATP sulfurase in the presence of APS. The ATP reacts with luciferin in the presence of luciferase to produce oxyluciferin which produces photons of light
What is high-throughput sequencing?
a variety of fast, cheap methods to sequence genomes
What does APS stand for?
adenosine 5' phophosulfate
What is the role of the DNA polymerase in pyrosequencing?
it catalyses the addition of a complimentary base pair from an activated nucleotide
What is the role of luciferase in pyrosequencing?
it catalyses the reaction between ATP and luciferin to form oxyluciferin
What is the role of ATP sulfurase in pyrosequencing?
it converts PPi to ATP in the presence of APS
What is the role of PPi in pyrosequencing?
it is converted to ATP by ATP sulfurase in the presence of APS
What is the role of APS in pyrosequencing?
it must be present for ATP sulfurase to catalyse PPi to ATP
What is the role of oxyluciferin in pyrosequencing?
it produces photons of light
What is the role of luciferin in pyrosequencing?
it reacts with ATP in the presence of luciferase to produce oxyluciferin
What is the role of ATP in pyrosequencing?
it reacts with luciferin in the presence of luciferase to produce oxyluciferin
What is the role of apyrase in pyrosequencing?
it removes left over bases from the reaction
Give an example of high throughput sequencing:
pyrosequencing
Give all the ingredients used in pyrosequencing:
single stranded DNA, primer, 4 enzymes, 2 substrates, 4 activated nucleotides
How does pyrosequencing methods compute the photons of light produced?
the light has different frequencies and single, double and triple peaks can form