Pyrosequencing reaction

Fig. 1. The Pyrosequencing reaction. A modified ATP is used for the nucleotide dispensations to prevent its direct nse by luciferase in the reaction. Modified and published with permission from Biotage AB.

Template preparation for pyrosequencing is straightforward. After generation of the template by PCR, the product should be purified prior to pyrosequencing. Unincorporated nucleotides and PCR primers in PCR reaction perturb the pyrosequencing reaction, and the salt in the PCR reaction can inhibit the enzyme system and should be removed or diluted. Depending on the sequence of the DNA template, up to 200 bp can be read. Stretches of three or more identical bases can lead to problems in data interpretation. As for every sequencing reaction, the primer should bind only once at the template. 

The technology company 454 Life sciences has developed a solid-phase parallel microarray system of microfluidic wells in which 10 oligonucleotide molecules per square centimeter are used to capture complementary DNA fragments. Pyrosequencing reactions are simultaneously monitored on each... 

It should be possible to develop either biochemical- or molecular-based tests for resistance in flatworms, as has been successfully used to detect insecticide resistance in insects. However, the necessary information is not available on the mechanism of resistance to be able to design tests of this type for use with trematodes or cestodes. When developed they will hopefully be based on real-time polymerase chain reaction (PCR) or pyrosequencing. 

An alternative method of DNA sequencing, called Pyrosequencing (3) uses a series of enzymatic reactions to enable the continuous readout of short stretches of DNA sequence without the requirement for electrophoretic fragment separation. The method may be useful for low-throughput research and clinical diagnostic applications, but is unlikely to be applied to high-throughput applications. 

Pyrosequencing is a method to determine the nucleic acid sequence of short segments without the use of electrophoresis. A sequencing primer is hybridized to a single-stranded template that is usually generated by PCR. Four enzymes, a DNA polymerase, ATP sulfurylase, luciferase and apyrase, and two substrates— adenosine 5 phosphosulfate and luciferin— are included in the reaction mixture (Figure 37-17). One of the four dNTPs is added to the reaction (dATPaS is substituted for dATP because it is incorporated by the polymerase but is not a luciferase substrate). If the base is complementary to the template strand, DNA polymerase catalyzes its incorporation. Each incorporation event is accompanied by release of a pyrophosphate (PPi) so that the quantity of PPi produced is equimolar to the... 

Dideoxynucleotides are commercially available with different fluorescent tags therefore, the resulting extension products can be examined using laser scanners to see which fluorophore has been incorporated. This method directly reveals the genotype of the template DNA. The method can be multiplexed and is commercially available (SNaPshot, see below). Alternatively to the use of dideoxynucleotides, regular deoxynucleotides can be used sequentially in the extension reaction. Here, the primer is essentially extended as in a regular PCR reaction. The only difference is the sequential addition of one nucleotide at a time. This method is commercially available (see pyrosequencing, described below). 

Pyrosequencing Primer extension Homogeneous reaction or solid support Luminescence 218,219,220,221,222... 

Pyrosequencing produces specific sequence data in the form of peaks on a pyrogram. It does not require the presence of a restriction enzyme site, and polymerase chain reaction (PCR) product and internal primer sites can vary in size and position. In addition, it can be used to identify tri-allelic, indel, and short-... 

Pyrosequencing is based on sequencing by synthesis. The assay takes advantage of the natural release of pyrophosphate whenever a nucleotide is incorporated onto an open 3 DNA strand. The released p5u-ophosphate is used in a sulfurylase reaction releasing ATP. The released ATP can be used by luciferase in the conversion of luciferin to oxyluciferin. The reaction results in the emission of light, which is collected by a CCD camera and recorded in the form of peaks, known as pyrograms (Fig. 1). When a nucleotide is not incorporated... 

A 20-pL PCR is ideal for Pyrosequencing however, if the PCR product is especially strong or wide-peak pyrograms occur, a 10-pL reaction will work well. For a 20-pL reaction based on ABl Amplitaq Gold PCR mastermix (Applied Biosystems, Foster City, CA) ... 

Fig. 4. The illustration of the reverse terminator sequencing and pyrosequencing. For reverse terminator sequencing, different dNTP is labelled with different fluorescence dye. Upwn addition of each dNTP, the reaction halts and the fluorescence is the recorded. Then the terminator and fluorescence dye of dNTP is cleaved. Subsequently, the next dNTP is incorporated and the whole process is iterated. For pyrosequendng, one single dNTP flows through with the addition of the nucleotide in the corresponding position. The release of the pyrophosphate will undergo enzymatic reaction to produce light. Therefore, the camera will record which of the fragments has this dNTP at its current position.

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