Pyrosequencing

Question 1

General overview of pyrosequencing

Answer 1

A method of “sequencing by synthesis”

i.e. taking a single strand of the DNA to be sequenced and synthesising its complementary strand enzymatically

Question 2

What is in the pyrosequencing pot?

Answer 2

ssDNA template to be sequenced hybridised to a sequencing primer
DNA polymerase
ATP sulfurylase
Luciferase
Luciferin
Apyrase

Question 3

General procedure for pyrosequencing

Answer 3

1. dNTPs are added stepwise to the mixture of ssDNA/enzymes
2. DNA polymerase incorporates the correct dNTP that is complementary to the template ssDNA strand
3. This dNTP incorporation releases pyrophosphate (PPi) in equal molarity to that of the incorporated nucleotide
4. PPi is converted into ATP by ATP sulfurylase
5. This ATP then acts as a substrate for the luciferase-catalysed conversion of luciferin into oxyluciferin, which produces visible light
6. The amount of light generated is estimated using a luminometer or CCD camera
7. The unincorporated dNTPs and excess ATP are degraded between each cycle by apyrase into dNMP and 2 Pi
8. The reaction can then restart with another nucleotide

Question 4

Limitations of pyrosequencing

Answer 4

No proof-reading activity limits the accuracy of the method
Difficult for sequences containing a large amount of repetitive DNA due to the non-linear light response following the incorporation of more than 3 or 4 identical nucleotides

Question 5

How is the ssDNA for pyrosequencing obtained?

Answer 5

By biotin capture on magnetic beads

Question 6

Required properties of the nucleotide-degrading enzyme apyrase

Answer 6

The enzyme must:
Hydrolyse all dNTPs at approx. the same rate, including the alpha-thio-dATP
Hydrolyse ATP to prevent its accumulation between cycles
Degrade nucleotides at a slower rate than they are incorporated into the strand by DNA polymerase - the two enzymes compete for the same substrate
Degrade ATP at a slower rate than ATP is synthesised by sulfurylase in order to obtain ATP concentrations and light production in proportion to the amount of PPi released

Question 7

Why is alpha-thio-dATP used instead of dATP?

Answer 7

To improve background/noise

Question 8

What happens to the activity of apyrase in later pyrosequencing cycles?

Answer 8

Activity is decreased

Due to accumulation of intermediate products (e.g. dNDP) and eventually undegraded dNTP

Question 9

How can the efficiency of apyrase in later cycles be improved?

Answer 9

By removing the intermediate nucleotide products by enzymes such as non-specific nucleoside diphosphatases
This will allow for longer reads

Question 10

Notes on pyrosequencing

Answer 10

Can be automated
Can be done in parallel
Can be done in real time
The process is extremely fast (approx. 3 seconds from start to finish) - 25 million nucleotides can be incorporated in 4 hours, which is 150x faster than conventional Sanger sequencing)

Question 11

Sanger sequencing

Answer 11

REVISE