Pyrosequencing methods

Fig. 3-41 Schematic depiction of the pyrosequencing method for determining the sequence of deoxynucieotides in a sampie of one form of DNA.

Delgado S, Cabrera-Rubio R, Mira A, Suarez A, Mayo B. Microbiological survey of the human gastric ecosystem using culturing and pyrosequencing methods. Microb Ecol. 2013 65 763-772. 

Van Booven, D. J. (2006) Pyrosequencing genotype storage techniques. Methods Mol Biol. 373, 177-186. 

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... 

A variety of molecular techniques are available to detect point mutations at a specific hot spot (e.g., the KRAS mutation at codons 12 and 13 or the BRAF mutation at codon 600). These methods include realtime PCR amplification and post-PCR melting curve analysis, allele-specific PCR, direct DNA sequencing, pyrosequencing, and PCR-RFLP. o,31-37 q methods demonstrate reliable detection of point mutations, such as the KRAS mutation in colorectal cancer or the BRAF mutation in thyroid cancer. 

Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. Mol Diagn. 2005 7 413-421. 

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). 

Nordstrom T, Nourizad K, Ronaghi M, etal. Method enabling pyrosequencing on double-stranded DNA. Anal Biochem 2000 282 186-193. 

This protocol assumes the use of a streptavidin/Sepharose bead set-up for Pyrosequencing on a hsPSQ96 or a hsPSQ96A system. The magnetic bead processing method for the PSQ96 or PSQ96MA is described elsewhere (8). 

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