Method Development and Applications of Pyrosequencing Technology

The ability to determine nucleic acid sequences is one of the most important platforms for the detailed study of biological systems. Pyrosequencing technology is a relatively novel DNA sequencing technique with multifaceted unique characteristics, adjustable to different strategies, formats and instrumentations. The aims of this thesis were to improve the chemistry of the Pyrosequencing technique for increased read-length, enhance the general sequence quality and improve the sequencing performance for challenging templates. Improved chemistry would enable Pyrosequencing technique to be used for numerous applications with inherent advantages in accuracy, flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted to sequencing short stretches of DNA. The major limiting factor was presence of an isomer of dATPaS, a substitute for the natural dATP, which inhibited enzyme activity in the Pyrosequencing chemistry. By removing this non-functional nucleotide, we were able to achieve DNA read-lengths of up to one hundred bases, which has been a substantial accomplishment for performance of different applications. Furthermore, the use of a new polymerase, called Sequenase, has enabled sequencing of homopolymeric T-regions, which are challenging for the traditional Klenow polymerase. Sequenase has markedly made possible sequencing of such templates with synchronized extension. The improved read-length and chemistry has enabled additional applications, which were not possible previously. DNA sequencing is the gold standard method for microbial and vial typing. We have utilized Pyrosequencing technology for accurate typing of human papillomaviruses, and bacterial and fungal identification with promising results. Furthermore, DNA sequencing technologies are not capable of typing of a sample harboring a multitude of species/types or unspecific amplification products. We have addressed the problem of multiple infections/variants present in a clinical sample by a new versatile method. The multiple sequencing primer method is suited for detection and typing of samples harboring different clinically important types/species (multiple infections) and unspecific amplifications, which eliminates the need for nested PCR, stringent PCR conditions and cloning. Furthermore, the method has proved to be useful for samples containing subdominant types/species, and samples with low PCR yield, which avoids reperforming unsuccessful PCRs. We also introduce the sequence pattern recognition when there is a plurality of genotypes in the sample, which facilitates typing of more than one target DNA in the sample. Moreover, target specific sequencing primers could be easily tailored and adapted according to the desired applications or clinical settings based on regional prevalence of microorganisms and viruses. Pyrosequencing technology has also been used for clone-checking by using preprogrammed nucleotide addition order, EST sequencing and SNP analysis, yielding accurate and reliable results.