Pyrosquencing

Question 1

Pyrosequencing

Answer 1

• 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
• no gels, fluorescent dyes, or ddNTPs

Question 2

Name 4 enzymes always present in pyrosequencing

Answer 2

1. DNA Polymerase
2. ATP sulfurylase
3. Luciferase
4. Apyrase

Question 3

Describe 3 primers used in pyrosequencing

Answer 3

1. Forward primer
2. Sequencing primer (downstream of forward primer, but upstream the target sequence)
3. Biotinylated reverse primer

Question 4

Describe sample prep for pyrosequencing (5)

Answer 4

1. PCR with forward and (biotinylated) reverse primer
2. Immobilize biotinylated PCR products onto streptavidin-coated beads
3. Separate strands (denatured by NaOH)
4. Wash/ neutralize immobilized strand
5. Anneal sequencing primer

Question 5

Every time a dNTP is added, a __ is removed

Answer 5

Every time a dNTP is added, a pyrophosphate is removed

Question 6

How is light detected in pyrosequencing ?

Answer 6

1. ATP generation:
   - dNTP added = pyrophosphate is removed
   - pyrophosphate and APS uses ATP-sulfurylase = ATP
2. Light generation:
   - ATP and luciferin uses Luciferase = light (oxyluciferin) correlates to 1 of 4 nucleotides
3. Reset:
   Apyrase degrades free dNTP and dATP for next round

NOTE: APS - Adenosine phosphosulfate

Question 7

Bisulfite DNA sequencing

Answer 7

• “methylation-specific sequencing”
• modification of chain termination sequencing/ pyrosequencing designed to detect methylated cytosines

Question 8

Methylated DNA is involved in (4):

Answer 8

• gene expression regulation
• chromatin structure regulation
• cell differentiation
• implicated in a number of diseases ie. cancer

Question 9

Describe Bisulfite DNA Sequencing Procedure (3)

Answer 9

1. cut genomic DNA with restriction enzymes (want shorter)
2. Gel electrophoresis = fragment of interest is purified from gel
3. DNA is denatured by heat and exposed to Bisulfite solution
   = deamination of cytosines TO URACILS = sequenced TO TYROSINE (bc using opposite strand) in PCR

NOTE: 5-methylated cytosines are unchanged; resistant to bisulfate

Question 10

Why can’t we use PCR products for Bisulfate treatment ?

Answer 10

No longer methylated

Question 11

Uses of pyrosequencing

Answer 11

• shorter sequence analysis (mutations or single nucleotide polymorphisms)
• infectious disease typing and HLA typing
• used in bisulfate sequencing