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

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 steps of pyrosequencing (5)

Answer 4

1. PCR with one of biotinylated primers
2. Immobilize biotinylated PCR products onto streptavidin-coated beads
3. Separate bands (denatured by NaOH)
4. Wash/ neutralize immobilized strand
5. Anneal sequencing primer

Question 5

Every time a dNTP is added, __ is removed

Answer 5

Every time a dNTP is added, *a pyrophosphate** is removed

Question 6

How is light generated 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)

Question 7

Bisulfite DNA sequencing

Answer 7

• “methylation-specific sequencing”
• modification of chain termination sequencing designed to detect methylated nucleotides

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

Described Bisulfite DNA Sequencing Procedure

Answer 9

• 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

Question 10

Why can’t we use PCR product ?

Answer 10

Not methylated