how does pyrosequencing singal work
chemiluminescnce - light signal when dNTP is added
four enzymes that are present at all times
DNA pol, ATP sulfurylase, Luciferase, Apyrase
how are dNTPs added and measured
- add dNTP (one at a time)
- clean up
- measure signal
- add dNTP…
What are the three primers added
forward
sequencing
reverse primer
where is the sequencing primer found
bt forward primer and target sequence
T/F reverse primer is biotinylated
T
what are the steps in pyrosequencing
- PCR with one primers biotinylated
- immobilize biotinylated PCR onto strepavidin beads
- denaturation in NaOH
- wash
- anneal sequecncing primers
what happens in both pyrosequencing and Sanger PCR when a dNTP is added onto the chain
pyrophosphate is lost to start the cascade
what is the pyrosequncing workflow
- extraction
2.PCR - sample prep
- Pyrosequecing
- analysis
What are the 5 steps in pyrosequencing
- dNTP is turned into DNA and PP (pyrophosphate) using polymerase
- APS + PP turns into ATP with sulfurylase
- ATP with Luciferin turns into oxiluciferin and light (signal) with luciferase
- Apyrase will remove ATP and junk nNTP
what is the device to detect light called
luminometer
what is pyrosequencing used for (4)
- short sequence analysis
- single nucleotide polymorphism
- HLA tpying
- BISULFITE SEQUENCING
What is the purpose of bisulfite sequencing
- genes cut with restritction enzymes
- agarose gel used
- DNA denaturation and exposure to bisulfite solution
- bisulfate incubation = deamination of UNmethylated cytosines into URACILS
why is genomic DNA used
we DONT use PCR product because it is NOT methylated
How would be untreated strand turn into a primer sequence
untreated sequence ->
bisulfite sequence ->
compliment ->
primer sequence
what template is used for PCR amplification
bisulfate treated templates
how is methylation detected
comparing a treated seuqence and untreated sequence
