454 pyrosequencing @ujjwalsirohi

454 pyrpsequencing

1. 454 PYROSEQUENCING
Roche sequencing
@ujjwalsirohi
Ph.Dscholar

2. 454 pyrosequencing
Pyrosequencingis a method of DNA sequencing
based on the "sequencing by synthesis"principle.
It relies on the detection of pyrophosphate
release on nucleotide incorporation.
454 pyrosequencingutilizes a single strand of DNA
with a length of 400-500bp.
Can sequence Mbsof DNA in ~$100

3. methodology
Sample preparation
Emulsion PCR
Alkaline degradation of PCR product
Beads loaded into wells of titanium covered plate
with luciferase, sulphurylase, primers, polymerase

4. The PyrosequencingEnzyme Cascade
Pyrosequencingrequires a single-stranded PCR ampliconthat serves as
DNA template, four different enzymes including DNA polymerase,
ATP sulfurylase, luciferase, and apyrase, and two different substrates
including adenosine 5′phosphosulfate(APS) and luciferin.
First, a sequencing primer is annealed to a single-stranded DNA
(ssDNA) template.
Upon addition of a single nucleotide, the DNA polymerase
incorporates the dNTPinto the growing strand, releasing
pyrophosphate (PPi).
ATP sulfurylasethen generates ATP from the PPiand substrate APS,
which activates luciferase-mediated conversion of luciferinto the light-
emitting oxyluciferin. Light is given off proportionate to the amount of
nucleotide added to the elongating strand and recorded by an inbuilt
CCD camera.
Excess nucleotide is degraded by apyrase, after which the next
nucleotide is dispensed. Comparing the peak light emission of
incorporation of C or T at a CpGsite within the amplicongives a precise
measure of the amount of methylationat that position within the
sample.

5. ePCR

7. Blank read