5.12:_Pyrosequencing

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<p class="lt-bio-4760" style="background-color: unset;">In laboratories around the world there is an intense desire to sequence more genomes.</p> <ul> <li class="lt-bio-4760" style="background-color: unset;">those of a wide variety of organisms to aid in establishing evolutionary relationships;</li> <li class="lt-bio-4760" style="background-color: unset;">those of pooled populations of microorganisms in, for examples, sea water, soil, the large intestine;</li> <li class="lt-bio-4760" style="background-color: unset;">other humans to look for genes that predispose to disease and genetic patterns in various ethnic groups.</li>
</ul> <p class="lt-bio-4760" style="background-color: unset;">All of the sequenced genomes listed in Genome Sizes were determined using the dideoxy method invented by Frederick Sanger and described elsehwere. However, now a great effort is being expended to find ways to sequence DNA more rapidly (and more cheaply).</p> <span id="The_Genome_Sequencer"></span><span id="The_Genome_Sequencer-2"></span><h2 style="background-color: unset;" class="lt-bio-4760">The Genome Sequencer</h2> <p class="lt-bio-4760" style="background-color: unset;">Several new methods are being developed and one is already commercially available (the Genome Sequencer 20 System). Its method is called pyrosequencing or sequencing by synthesis. It works like this.</p> <ul> <li class="lt-bio-4760" style="background-color: unset;">The DNA to be sequenced is broken up into fragments of ~100 base pairs and denatured to form single-stranded DNA (ssDNA).</li> <li class="lt-bio-4760" style="background-color: unset;">Single ssDNA fragments are attached to microscopic beads, which are separated from each other.</li> <li class="lt-bio-4760" style="background-color: unset;">The polymerase chain reaction (PCR) is run on each bead so that each becomes coated with ~ 10 million identical copies of that fragment.</li> <li class="lt-bio-4760" style="background-color: unset;">The beads are placed singly into separate, microscopic wells (~200,000 of them).</li> <li class="lt-bio-4760" style="background-color: unset;">Each well receives a cocktail of reagents: <ul> <li class="lt-bio-4760" style="background-color: unset;">DNA polymerase &mdash; for adding deoxyribonucleotides to the ssDNA</li> <li class="lt-bio-4760" style="background-color: unset;">adenosine phosphosulfate (APS)</li> <li class="lt-bio-4760" style="background-color: unset;">ATP sulfurylase &mdash; an enzyme that forms ATP from adenosine phosphosulfate (APS) and pyrophosphate (PPi)</li> <li class="lt-bio-4760" style="background-color: unset;">luciferin</li> <li class="lt-bio-4760" style="background-color: unset;">luciferase - an ATPase that catalyzes the conversion of luciferin to oxyluciferin with the liberation of light</li> </ul> </li>
</ul> <figure><img style="width: 240px; height: 230px;" class="internal" alt="Pyrosequencing diagram: dGTP added to a bead-bound DNA template releases PPi, converted to ATP, driving luciferase light emission." loading="lazy" width="240px" height="230px" src="https://bio.libretexts.org/@api/deki/files/6533/pyro1.png?revision=1&amp;size=bestfit&amp;width=240&amp;height=230" aria-describedby="remedy-description-cd3c1626777b39bf" /><span id="remedy-description-cd3c1626777b39bf" data-remedy-image-description="cd3c1626777b39bf" style="display: block; white-space: pre-line;">Flowchart of the pyrosequencing reaction (sequencing by synthesis) in a single well. At upper left, a gray circle represents a bead carrying a single-stranded DNA template. The template's visible sequence reads G G A A C T A C from the bead outward, ending in a segmented box representing the remaining, unshown portion of the fragment. Above the template, an arrow labeled 'Direction of synthesis' points left, toward the bead, and a solid bar depicts the newly synthesized complementary strand; the letter G marks the nucleotide just added, paired with the template C. The incoming nucleotide, dGTP, is incorporated into the strand, and the pathway branches downward to release pyrophosphate (PPi). PPi plus adenosine phosphosulfate (APS) is converted by the enzyme ATP sulfurylase (labeled in italics) into ATP. ATP then combines with luciferin, and the enzyme luciferase (italics) catalyzes conversion to oxyluciferin with emission of light, depicted as a green oval labeled 'Light'. Downward arrows connect each step in a vertical cascade from dGTP to light, illustrating how each nucleotide addition is coupled, via PPi and ATP production, to a detectable light signal.</span>                    <img style="width: 240px; height: 212px;" class="internal" alt="Pyrogram from a pyrosequencing run: light peaks (0???4) per nucleotide flow in T,A,C,G cycles; template T CCC AT CAA GG A T CC TGCCC above." loading="lazy" width="240px" height="212px" src="https://bio.libretexts.org/@api/deki/files/6534/pyro2a.png?revision=1&amp;size=bestfit&amp;width=240&amp;height=212" aria-describedby="remedy-description-0f98e6113045a95f" /><span id="remedy-description-0f98e6113045a95f" data-remedy-image-description="0f98e6113045a95f" style="display: block; white-space: pre-line;">Bar chart (pyrogram) illustrating Figure 5.12.1, 'Pyrosequencing run and the data produced by a single well.' The vertical axis is unlabeled but scaled 0 to 4 in unit steps; it represents light intensity, which is proportional to the number of identical nucleotides incorporated during a flow. The horizontal axis shows the order of nucleotide addition, repeating the cycle T, A, C, G for five cycles (20 positions). Above the chart, the deduced template sequence is printed: T CCC AT CAA GG A T CC TGCCC. Peak heights (number of nucleotides added) by cycle: cycle 1 ??? T: tiny near-zero tick, A: 1, C: tiny tick, G: 3; cycle 2 ??? T: 1, A: 1, C: tiny tick, G: 1; cycle 3 ??? T: 2, A: tiny tick, C: 2, G: tiny tick; cycle 4 ??? T: 1, A: 1, C: tiny tick, G: 2; cycle 5 ??? T: tiny tick, A: 1, C: 1, G: 3. The tiny near-baseline ticks indicate a nucleotide was flowed but none was incorporated. Read in order, the peaks give the synthesized strand A-GGG-T-A-G-TT-CC-T-A-GG-A-C-GGG, which is complementary to the template sequence displayed above the chart.</span><figcaption>Figure 5.12.1 Pyrosequencing run and the data produced by a single well</figcaption></figure> <p class="lt-bio-4760" style="background-color: unset;">The sequencing run:</p> <ul> <li class="lt-bio-4760" style="background-color: unset;">Each of the thousands of wells is flooded with one four deoxyribonucleotides, dTTP, dCTP, and dGTP, but instead of dATP (which would trigger the luciferin reaction), deoxyadenosine alpha-thiotriphosphate (dATP&alpha;S) is used instead. DNA polymerase ignores the difference and uses it whenever a T is encountered on the ssDNA template, but luciferase doesn't recognize to it.</li> <li class="lt-bio-4760" style="background-color: unset;">In any well where the complementary nucleotide is present at the 3' end of the template, the nucleotide is added and pyrophosphate is liberated.</li> <li class="lt-bio-4760" style="background-color: unset;">The amount of light is proportional to the number of that nucleotide added. So if, for example, the incoming nucleotide is dGTP, and there is a string of 3 Cs on the template, the light emitted will be 3 times brighter than if only one C is present.</li> <li class="lt-bio-4760" style="background-color: unset;">A detector picks up the light (if any) from each well and the data are recorded.</li> <li class="lt-bio-4760" style="background-color: unset;">Then each of the remaining 3 nucleotides are added in sequence.</li> <li class="lt-bio-4760" style="background-color: unset;">Then the sequence of 4 additions is repeated until synthesis is complete.</li>
</ul> <p class="lt-bio-4760" style="background-color: unset;">The above diagram also shows the type of data produced in a single well. The height of the peak of light production gives the number of additions that occurred when a particular nucleotide was added (bottom). Computer software then displays the template sequence (top) for each of the thousands of different fragments sequenced. With this technology, as many as 20 million base pairs of genome sequence can be learned in an instrument run of less than 6 hours.</p>

<hr class="autoattribution-divider" /><div class="autoattribution"><p>This page titled <a target="_blank" class="internal mt-self-link" href="/Sandboxes/johnnyphung/biology/05:_DNA/5.12:_Pyrosequencing">5.12: Pyrosequencing</a> is shared under a <a rel="nofollow" href="https://creativecommons.org/licenses/by/3.0" target="_blank">CC BY  3.0 (creativecommons.org)</a>  license and was authored, remixed, and/or curated by <a rel="nofollow" target="_blank" href="http://www.biology-pages.info/">John W. Kimball (biology-pages.info)</a> via <a rel="nofollow" href="https://www.biology-pages.info/" target="_blank">source content (biology-pages.info)</a> that was edited to the style and standards of the LibreTexts platform.</p></div>

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