PYROSEQUENCING- PRINCIPLES AND APPLICATIONS

Life Science-Molecular Biology

Abstract

Pyrosequencing is the first alternative to the conventional Sanger method for de novo DNA sequencing.
Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. It
employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis.
Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily
automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and
gel-electrophoresis. Pyrosequencing has opened up new possibilities for performing sequence-based DNA
analysis. The method has been proven highly suitable for single nucleotide polymorphism analysis and
sequencing of short stretches of DNA. Pyrosequencing has been successful for both confirmatory
sequencing and de novo sequencing. By increasing the read length to higher scores and by shortening the
sequence reaction time per base calling, pyrosequencing may take over many broad areas of DNA
sequencing applications as the trend is directed to analysis of fewer amounts of specimens and large-scale
settings, with higher throughput and lower cost. This article considers key features regarding different
aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing
modes, instrumentation, limitations, potential and future applications.