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Optical sequencing: A new approach ...

Ramanathan, Arvind.

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Optical sequencing: A new approach for single DNA molecule sequencing.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Optical sequencing: A new approach for single DNA molecule sequencing./
作者:	Ramanathan, Arvind.
面頁冊數:	192 p.
附註:	Adviser: David C. Schwartz.
Contained By:	Dissertation Abstracts International63-04B.
標題:	Chemistry, Analytical. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3048861
ISBN:	0493632077

Optical sequencing: A new approach for single DNA molecule sequencing.
Ramanathan, Arvind.

Optical sequencing: A new approach for single DNA molecule sequencing. - 192 p.

Adviser: David C. Schwartz.

Thesis (Ph.D.)--New York University, 2002.

Optical sequencing is a new approach for sequencing single DNA molecules. This takes advantage of the principles established in the optical mapping system. Optical sequencing involves the DNA polymerase mediated incorporation of fluorochrome labeled nucleotides (FdNTP) into single DNA molecules elongated on optical mapping surfaces. Each optical sequencing cycle include a single nucleotide incorporation, optical detection of a small number of fluorochromes followed by photobleaching. The development of this approach involves:

ISBN: 0493632077Subjects--Topical Terms:

586156
Chemistry, Analytical.

Optical sequencing: A new approach for single DNA molecule sequencing.
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502 $a Thesis (Ph.D.)--New York University, 2002.
520 $a Optical sequencing is a new approach for sequencing single DNA molecules. This takes advantage of the principles established in the optical mapping system. Optical sequencing involves the DNA polymerase mediated incorporation of fluorochrome labeled nucleotides (FdNTP) into single DNA molecules elongated on optical mapping surfaces. Each optical sequencing cycle include a single nucleotide incorporation, optical detection of a small number of fluorochromes followed by photobleaching. The development of this approach involves:
520 $a Studying the effect of consecutive incorporations of FdNTPs, on DNA polymerase action. A polyacrylamide gel based assay was used to study the effect of different fluorochromes, DNA polymerases, buffers and linker lengths between the fluorochrome and nucleotide, on consecutive incorporations. Molecular modeling studies indicated that, with a longer linker length and an appropriate fluorochrome and DNA polymerase, consecutive FdNTP incorporations could be maximized. 40 consecutive incorporations were observed, by using sequenase v 2.0 and fluorescein with a 12 carbon atom linker, in the presence of manganese buffer. It was also confirmed that fluorochrome bleaching did not have a deleterious effect on subsequent DNA polymerase action.
520 $a The detection and quantitation of single flurochromes. A DNA target with a known distribution of R110 dUTPs was used for the photodetection of single fluorochromes. The point spread function of the optics, the Gaussian profile, photobleaching characteristics, and colocalization with a DNA backbone, were used as evidence for single fluorochrome detection. The normalized intensity of the Gaussian profiles was used to quantitate single fluorochromes. The action of sequenase v 2.0 on surface elongated DNA was studied by the incorporation and quantitation of FdNTPs, on randomly gapped single lambda DNA molecules. An optical sequencing cycle was also demonstrated, by incorporation of R110 dUTPs and R110 dCTPs into the gapped template. This was followed by the incorporation and detection of a single R110 dUTP on a surface fixed DNA template of a known sequence. Finally an optical sequencing cycle on this construct was demonstrated as a proof of principle of the optical sequence approach.
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