Pyrosequencing Flashcards
What is the main hindrance of Sanger sequencing?
They require a DNA size determination step
What is the issue with Sanger sequencing requiring a size determination step?
It constrains the number of DNA clones that can be sequenced in a single sequencing run
What was the early aim of NGS development?
To come up with a method that relied only upon light detection
What is the first step of pyrosequencing?
Clonal amplification of DNA fragments
What is the first stage of clonal amplification?
Fragmenting the genome into tiny pieces
What is ligated onto the ends of the tiny fragments of DNA?
Adaptors
What are adaptors in clonal amplification?
Bits of DNA of known sequence that you can design primers against
What does mixing water and oil together result in?
Water droplets suspended in oil
How many water droplets are there?
Far more than the number of DNA molecules
Why are there so many water droplets in clonal amplification?
So only a single DNA fragment will enter each droplet
What added to each droplet once the DNA fragment is in there?
PCR equipment–> polymerase, buffers, primers (complementary to adaptors)
What does each water bead act as?
A microreactor
How does each water droplet act as a microreactor?
One PCR reaction (for each DNA fragment) occurs in each droplet
What are the beads of water spread onto once the PCR is completed
A pico-titer plate
What is on pico-titer plates?
A million wells per plate, each that can hold one water droplet
Where do the water droplets go in the pico-titer plates?
Each droplet goes into one of the wells
What is the second step of pyrosequencing?
Pyrosequencing
Where is the pyrosequencing done?
In the pico-titer plates
First step of pyrosequencing?
One type of nucleotide is added to the wells at a time
What happens when the correct nucleotide is added (i.e. one that is complementary)?
Pyrophosphate is released
Why is pyrophosphate released when the correct nucleotide is added?
It is a byproduct of incorporating a nucleotide into DNA
What is pyrophosphate a substrate for?
Sulfurylase enzyme
What does sulfurylase do to pyrophosphate?
Converts it into ATP
What is done with the ATP made from pyrophosphate?
Luciferase uses it to generate a flash of light
What does a flash of light during pyrosequencing indicate?
A successful addition of a nucleotide
What is added after each nucleotide is added in pyrosequencing?
Apysase enzyme
Why is apyrase enzyme added after each nucleotide?
To degrade the nucleotide so it doesnt confuse the machine
Why may the intensities of light produced by pyrosequencing be different?
Multiple of the same nucleotide may be added consecutively
What two bits of information about the flashes of light are required to determine the sequence in pyrosequencing?
The order of flashes, and their intensity
