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Pyrosequencing, Broken Down
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As our genome libraries and the need to conduct genetic screening continue to grow, newer and more innovative ways of sequencing also increase. One general term used for a more recently developed method is sequencing by synthesis. This is the method of sequencing a stretch of DNA by utilizing some sort of indicator whenever a particular nucleotide is added. More specifically, pyrosequencing is an even more recent development under the umbrella of sequencing by synthesis and is now being used in a wide range of labs.

The procedure for pyrosequencing is fairly straightforward, with the correct equipment, and can be summed up in five steps. The first step is to amplify the DNA being sequenced using PCR and incubate it with the proper enzymes. The list of pyrosequencing specific enzymes, in addition to the ones traditionally used for PCR include APS, ATP sulfurylase, luciferin, luciferase and apyrase. Step two involves the addition of a dNTP. If the dNTP is complimentary to the strand, then the reaction will cause one PPi to be released per dNTP being added.

In the third step, PPi is used to make ATP, by APS, in order to power the conversion of luciferon which is the process where the indicator light is released. This light is measured by a CCD chip in the program and a pyrogram is made depicting the number of dNTPs that were added through the use of peaks. In step four, the ATP and unused nucleotides need to be disposed of for the next group of dNTPs and thus are degraded by apyrase. The last step is mainly a summary of the steps above since it is to repeat them until the length of the DNA segment is sequenced, and the end result would be a complete pyrogram of the segment being examined.

Though the technique of pyrosequencing is extremely useful because of its high level of accuracy, there are a few areas where researchers should be called on to help make improvements. These include the length of the DNA segment that can be sequenced and the accuracy in the very beginning of the process. Segment lengths that can currently be sequenced, at a time, are 300-1000 base pairs long and tend to have questionable quality in approximately the first 30 base pairs. In order to make the process more efficient, we need to be able to sequence longer stretches of DNA at a time and with a more reliable starting position for the process. These improvements could really help to make the field of genomics more efficient and perhaps more available to make more improvements in modern medicine.


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