Newer NGS techniques Flashcards
Setup of Single molecule real time (SMRT)?
DNA polymerase is fixed to the bottom of a well
What is done to the well that polymerase is fixed to?
The small part that has the polymerase is iluminated
What is the illuminated part of the well called?
Zero-mode waveguide
Where are the DNTPs in SMRT?
Mobile in solution
What happens when the polymerase gets hold of the DNTP that is next in the nucleotide sequence?
A flash of light is given off
Why is a flash of light given off when the DNTP is incorperated into the DNA?
It is held in the zero-mode waveguide long enough to be excited
Where on the nucleotide in SMRT is the fluorescent group added?
The phosphate group of the nucleotide
What happens to the fluorescent group when the DNTP is incorporated into the polymerase?
It is released and floats off into solution
Why does the fluorescent group of the DNTP not constantly give off flashes of light?
They dont stay in the zero-mode waveguide for long enough
What does nanopore sequencing rely on?
Ion channels found in cell membranes
What are ion channels used for in nanopore sequencing?
DNA synthesis
What are the ion channels embedded into in nanopore sequencing?
an inert membrane
What is the inert membrane bathed in in nanopore sequencing?
An electrolyte solution
What is applied across the membrane in nanopore sequencing?
Potential difference
What does applying a potential difference across the membrane do in nanopore sequencing?
Causes ions to flow through the pore–> steady current is recorded
What happens to the current if something enters the pore?
It would reduce as the flow of ions would decrease
What is complexed to the DNA?
Motor enzyme
Where does the motor enzyme go?
On the pore
What do the nucleotides do as they are being added to the double helix?
Partially block the pore
What does the blockage of the pore do to the current?
Causes it to deflect
What is specific about each nucleotides effect on the current?
Each one will give the same effect every time e.g. A=0.9, G=1.1 etc
