期刊
ANALYTICAL BIOCHEMISTRY
卷 363, 期 2, 页码 275-287出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.02.002
关键词
pyrosequencing; sequencing-by-synthesis; enzyme kinetics; read-length; DNA sequencing; enzyme simulation
资金
- NHGRI NIH HHS [R01HG003571, P01 HG000205, P01HG00205, R01 HG003571, R01 HG003571-03] Funding Source: Medline
Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. In this study, we have characterized the main read length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read length of both systems based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized extension due mainly to a decrease in apyrase s efficiency in degrading excess nucleotides proves to be the main limiting factor of read length. Replacing apyrase with a washing step for removal of excess nucleotide proves to be essential in improving the read length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read length of Pyrosequencing would be well beyond 300 bases. (c) 2007 Elsevier Inc. All rights reserved.
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