Pyrosquencing Flashcards
1
Q
Pyrosequencing
A
- Principle: sequencing by synthesis
- stepwise synthesis of DNA; dNTPs added and cleaned up one at a time
- a light signal produced when a dNTP is added
2
Q
Name 4 enzymes always present in pyrosequencing
A
- DNA Polymerase
- ATP sulfurylase
- Luciferase
- Apyrase
3
Q
Describe 3 primers used in pyrosequencing
A
- Forward primer
- Sequencing primer downstream of forward primer, but upstream the target sequence
- Biotinylated reverse primer
4
Q
Describe steps of pyrosequencing (5)
A
- PCR with one of biotinylated primers
- Immobilize biotinylated PCR products onto streptavidin-coated beads
- Separate bands (denatured by NaOH)
- Wash/ neutralize immobilized strand
- Anneal sequencing primer
5
Q
Every time a dNTP is added, __ is removed
A
Every time a dNTP is added, *a pyrophosphate** is removed
6
Q
How is light generated in pyrosequencing ?
A
- ATP generation:
- dNTP added = pyrophosphate is removed
- pyrophosphate and APS uses ATP-sulfurylase = ATP - Light generation:
- ATP and luciferin uses Luciferase = light (oxyluciferin)
7
Q
Bisulfite DNA sequencing
A
- “methylation-specific sequencing”
- modification of chain termination sequencing designed to detect methylated nucleotides
8
Q
Methylated DNA is involved in (4):
A
- gene expression regulation
- chromatin structure regulation
- cell differentiation
- implicated in a number of diseases ie. cancer
9
Q
Described Bisulfite DNA Sequencing Procedure
A
- cut genomic DNA with restriction enzymes (want shorter)
- Run agarose gel = fragment of interest are purified from gel
- DNA is denatured by heat and exposed to Bisulfite solution
= deaminase on of cytosines TO URACILS = sequenced TO TYROSINE (bc using opposite strand)
NOTE: 5-methylated cytosines are unchanged
10
Q
Why can’t we use PCR product ?
A
Not methylated