Advanced Pharmaceutical Biotechnology Phar571/Lec. 4

Dr. Yara Al Tall

Pyrosequencing

Pyrosequencing is the second important type of DNA sequencing methodology that is in use today.

Pyrosequencing does not require electrophoresis or any other fragment separation procedure and so is more rapid than chain termination sequencing.

Pyrosequencing It is only able to generate up to 150 bp in a single

experiment, and at first glance might appear to be less useful than the chain termination method, especially if the objective is to sequence a genome.

The advantage with pyrosequencing is that it can be automated in a massively parallel manner that enables hundreds of thousands of sequences to be obtained at once, perhaps as much as 1000 Mb in a single run.

Pyrosequencing

Sequence is therefore produced much more quickly than is possible by the chain termination method.

This explains why pyrosequencing is gradually taking over as the method of choice for genome projects.

Pyrosequencing Pyrosequencing, like the chain termination

method, requires a preparation of identical single-stranded DNA molecules as the starting material.

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Pyrosequencing involves detection of pulses of chemiluminescence

1. Step 1, A sequencing primer is hydridized to a single-stranded DNA fragment that serves as a template.

Mixtures incubated with the enzymes; DNA polymerase, ATP sulfurylase, Luciferase, & apyrase.

PLUS Substrates which are, coenzymes adenosine 5’ phosphsulfate (APS), & luciferin

Pyrosequencing involves detection of pulses of chemiluminescence

2. The first deoxyribonucleotide triphosphate (dNTP) is added to the reaction.

DNA polymerase catalyzes the incorporation of the deoxyribonucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand

Pyrosequencing involves detection of pulses of chemiluminescence

Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

Pyrosequencing involves detection of pulses of chemiluminescence

3. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5’ phosphosulfate (APS).

Pyrosequencing involves detection of pulses of chemiluminescence

ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP.

Pyrosequencing involves detection of pulses of chemiluminescence

The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) chip and seen as a peak in the raw data output (Pyrogram).

The height of each peak (light signal) is proportional to the number of nucleotides incorporated.

Pyrosequencing involves detection of pulses of chemiluminescence

4. Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides & ATP.

When degradation is complete, another nucleotide is added.

Pyrosequencing involves detection of pulses of chemiluminescence

5. Addition of dNTPs is performed sequentially.

As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram trace.

Pyrosequencing involves detection of pulses of chemiluminescence

Pyrosequencing involves detection of pulses of chemiluminescence

Of course, if all four deoxynucleotides were added at once, then flashes of light would be seen all the time and no useful sequence information would be obtained This is why we add a nucleotidase enzyme (Apyrase), so degrade any unincorporated nucleotides (dNTP).

Massively parallel pyrosequencing The high throughput version of pyrosequencing

usually begins with genomic DNA.

The DNA is broken into fragments between 300 and 500 bp in length and each fragment is ligated to a pair of adaptors, one adaptor to either end.

Massively parallel pyrosequencing These adaptors play two important roles: First,

they enable the DNA fragments to be attached to small metallic beads.

This is because one of the adaptors has a biotin label attached to its 5 end, and the beads are ′coated with streptavidin, to which biotin binds with great affinity

Massively parallel pyrosequencing

Massively parallel pyrosequencing DNA fragments therefore become attached to

the beads via biotin-streptavidin linkages.

The ratio of DNA fragments to beads is set so that, on average, just one fragment becomes attached to each bead.

Massively parallel pyrosequencing Each DNA fragment will now be amplified by PCR

so that enough copies are made for sequencing.

The adaptors now play their second role as they provide the annealing sites for the primers for this PCR.

The same pair of primers can therefore be used for all the fragments, even though the fragments themselves have many different sequences.

Massively parallel pyrosequencing If the PCR is carried out immediately then all we will

obtain is a mixture of all the products, which will not enable us to obtain the individual sequences of each one.

To solve this problem, PCR is carried out in an oil emulsion, each bead residing in its own aqueous droplet within the emulsion.

Each droplet contains all the reagents needed for PCR, and is physically separated from all the other droplets by the barrier provided by the oil component of emulsion.

Massively parallel pyrosequencing

Massively parallel pyrosequencing After PCR, the aqueous droplets are transferred

into wells on a plastic strip so there is one droplet and hence one PCR product per well, and the pyrosequencing reactions are carried out in each well.

Additional reference to the text book

Mardis E.R. Annual Review of Genomics and Human Genetics 9:387-403 (2008)