DNA sequencing - lecture 15 Flashcards
how does Sanger sequencing work?
- DNA clones generated by PCR
- Clones sequences during polymerase - mediated synthesis.
- Random termination results in DNA fragments of varying sizes which can be analysed in determine nucleotide sequence
How are the nucleotides modified so that synthesis terminates ( in Sanger sequencing)?
- Differ in the 3 prime position, they have an H instead
- Polymerase requires the OH group, therefore terminates
ddNTP vs dNTP?
ddNTP is a modified nucleotide
How are the ddNTPs labelled?
Fluorescently labelled
How do you read the polyacrimide gel of the Sanger sequence?
- Determine the different fluorescent colours, Tells the sequence
What is illumina sequencing?
A ‘ Next - Generation - Sequencing’ platform
How does clonal bridge amplification work (part of the illuminati sequencing)
The library is flowed over the flow cell and DNA fragments with the P5 and P7 attachment sites will anneal to these sites on the flow cell.
These fragments are amplified resulting in covalently bound DNA fragments on the flow cell.
The original template is denatured and washed away.
Clusters of clonally amplified DNA fragments are formed in a process called bridge PCR.
The covalently bound DNA fragments will anneal to the neighbouring P5 or P7 attachment site and will form a bridge.
The fragment is then amplified and the resulting dsDNA denatured which results in two covalently bound DNA fragments on the surface of the flow cell.
Why is clonal bridge amplification useful?
- get millions of tiny fragments
Can quickly sequence the human genome in a few hours
What is reversible nucleotide made up of?
- Fluorophore group which illuminates light but is also cleavable
- A reversible terminato
what is the mechanism for using reversible dye chemistry?
- polymerase adds one modified nucleotide at a time
- Emits , light , camera records the slight
- chemicals added to remove fluorophore group and terminator group
- wash away chemicals, polymerase added next nucleotide
What is Singe Molecule Real Time sequencing?
- Enables DNA to sequence without any PCR amplification
- obtained in real time
- enables direct detection of DNA mutations
How does Single Molecule Real Time sequencing work?
- DNA polymerase attached to the bottom of a well
- DNA polymerase sits in a Zero - Mode Wave guide
- Each nucleotide fluorescently labelled and flashes when incorporated, then disappears
Where is the fluorophore attached to on the nucleotide, in Single Molecule Real Time Sequencing ?
Attached to the phosphate groups . When the disphosphate group cleaved, so if the F which floats off
Why is Single - molecule - Real - Time sequencing useful?
- Can identify mutations
- mutation makes the nucleotide a bit bulky , this hinders the rate which the DNA strand passes through the polymerase , delay fro the next nucleotide
What does ddNTP stand for?
Dideoxynucleotide
