Pyrosequencing-An Alternative to Traditional Sanger Sequencing

Problem statement: Pyrosequencing has the potential to rapidly and reliably sequence DNA taking advantages over traditional Sanger di-deoxy sequencing approach. Approach: A comprehensive review of the literature on the principles, applications, challenges and prospects of pyrosequencing was performed. Results: Pyrosequencing was 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 had the potential advantages of accuracy, flexibility, parallel processing and could be easily automated. The technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. Pyrosequencing had opened up new possibilities for performing sequence-based DNA analysis. The method had been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Pyrosequencing had 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 was directed to analysis of fewer amounts of specimens and large-scale settings, with higher throughput and lower cost. Conclusion/Recommendations: The Competitiveness of pyrosequencing with other sequencing methods can be improved in future."