Lecture 4 Flashcards
Sequencing by synthesis is
the most effective way to synthesis DNA
Sanger sequencing?
DNA clones are generated by a standard PCR reaction
Polymerase-mediated synthesis is critical to ensure
random terminations, resulting in Varying DNA fragments
dNTP vs ddNTP
deoxynucelotidetriosephosphate vs dideoxynucelotidetriophophate
What do ddNTP’s cause?
Termination
ddNTP are labelled?
Flourescently
Gel used in sanger sequencing
Polyacrylamide gel
How can we detect the sequence from a gel?
Fragment which travels the least= largest= first nucleotide which correlates to the colour.
Mechanical detection of colours of ddNTP’s
Laser beamer, photomultiplier to form an electro gram with the colours present.
Peak=termination
Illumina sequencing platform
Next generation
Illumina relies on
Dye terminator chemistry
BRIDGE AMPLIFICON?
found on glass slide and allows for clonal clusters to accumulate
Method of illumina sequencing.
1- DNA attaches to a flow cell
(a flow cell has multiple primers)
2- DNA forms a bridge with another primer
3- PCR HEAT UP, will continue to replicate and form a cluster
Reversible dye terminator chemistry: FUNCTION OF REVERSIBLE TERMINATOR
blocks the addition of another NT.
F group in reversible dye terminator chemistry
Chorophore group which is cleavable if the correct chemical Is added
When F cleaved
Light given off which can be detected by a camera
Advantage of Illumina sequencing
Multiple clusters are doing the same cloning at the same time therefore a quick process.
- Few hours
SMRT stands for
single molecule Real time sequencing
what kind of technique is SMRT?
Next generation
What does SMRT allow direct detection of?
DNA modifications.
Method of SMRT sequencing
1- Plate with wells in
2- DNA polymerase is attached to the bottom of the well
3- Zero-mode waveguide: which illuminates the proportion in which DNA polymerase is sat in
4- If the correct nucleotide approaches the DNA polymerase, it is held in the active site long enough for. flash of light to be given off, which is detected by a camera
Chemistry behind SMRT sequencing
Addition of a chlorophore group on dNTP (at end of tri phosphate) when diphosphate is cleaved light is given off
Delay in light given off indicates?
Hindered rate in which that nucleotide is passing through DNA polymerase, after the delay, a modification occurs
most widely used NGS platform?
ILLUMINA
