Abstract
As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bedside DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which cannot be easily sequenced by a conventional gel-based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC-rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kheterpal I, Mathies RA (1999) Capillary array electrophoresis DNA sequencing. Anal Chem 71:31A–37A
Venter JC et al (2001) The sequence of the human genome. Science 291:1304–1313
Ramsay G (1998) DNA chips: state-of-the art. Biotechnology 16:40–44
Fan JB, Chen X, Halushka MK, Berno A, Huang X, Ryder T, Lipshutz RJ, Lockhart DJ, Chakravarti A (2000) Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res 10:853–860
Kirpekar F, Nordhoff E, Larsen LK, Kristiansen K, Roepstorff P, Hillenkamp F (1998) DNA sequence analysis by MALDI mass spectrometry. Nucleic Acids Res 26:2554–2559
Chen J, Iannone MA, Li MS, Taylor JD, Rivers P, Nelsen AJ, Slentz-Kesler KA, Roses A, Weiner MP (2000) A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. Genome Res 10:549–557
Landegren U, Nilsson M, Kwok PY (1998) Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. Genome Res 8:769–776
Nollau P, Wagener C (1997) Methods for detection of point mutations: performance and quality assessment. Clin Chem 43:1114–1128
Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363–365
Hyman ED (1988) A new method of sequencing DNA. Anal Biochem 174:423–436
Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89
Nyrén P, Lundin A (1985) Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis. Anal Biochem 151:504–509
Szita N, Sutter R, Dual J, Buser R (2000) Proceedings of MEMS2000, Miyazaki Japan, January 23–27, 2000. pp 409–413
Ronaghi M, Nygren M, Lundeberg J, Nyren P (1999) Analyses of secondary structures in DNA by pyrosequencing. Anal Biochem 267:65–71
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhou, G., Kamahori, M., Okano, K., Harada, K., Kambara, H. (2016). Pyrosequencer Miniaturized with Capillaries to Deliver Deoxynucleotides. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3308-2_23
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3306-8
Online ISBN: 978-1-4939-3308-2
eBook Packages: Springer Protocols