Pyrosequencing : History, biochemistry and future

• Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.

Nyckelord

pyrosequencing

real-time monitoring

SNP

bioluminscence

dna-binding-protein

polymerase-chain-reaction

single-nucleotide polymorphisms

micromachined filter-chamber

allele-specific extension

real-time pyrophosphate

tumor-suppressor gene

i reaction pathway

firefly luciferase

escherichia-coli

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