Sequencing methods 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. 

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

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. 

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

In this chapter, the Maxam-Gilbert method and the Sanger method for ab initio sequencing of long nucleic acid chains are discussed. Two alternative methods for sequencing of shorter DNA strands are described in chapter 5 with DNA arrays, sequences of up to a few hundred base pairs can be determined (section 5.3), while pyrosequencing is capable of identifying sequences of up to 50 bp (section 5.4). 

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