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Development of methodology for the s...

Zabzdyr, Jennifer Lee.

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Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis./
作者:	Zabzdyr, Jennifer Lee.
面頁冊數:	145 p.
附註:	Source: Dissertation Abstracts International, Volume: 65-04, Section: B, page: 1842.
Contained By:	Dissertation Abstracts International65-04B.
標題:	Chemistry, Analytical. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3130287
ISBN:	0496775391

Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis.
Zabzdyr, Jennifer Lee.

Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis. - 145 p.

Source: Dissertation Abstracts International, Volume: 65-04, Section: B, page: 1842.

Thesis (Ph.D.)--University of California, Riverside, 2004.

The work presented herein describes the development of capillary electrophoresis (CE)-based methodology for the analysis of nucleic acids originating from individual cells. Three distinct approaches were used to accomplish this goal: (1) evaluation of nucleic acid dyes for DNA detection; (2) the study of DNA and RNA migration in the presence of electroosmotic flow; and (3) the development of a method to measure single-cell messenger RNA (mRNA) using the reverse transcriptase-polymerase chain reaction (RT-PCR).

ISBN: 0496775391Subjects--Topical Terms:

586156
Chemistry, Analytical.

Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis.
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100 1 $a Zabzdyr, Jennifer Lee. $3 1939195
245 1 0 $a Development of methodology for the study of nucleic acids from single cells using capillary electrophoresis.
300 $a 145 p.
500 $a Source: Dissertation Abstracts International, Volume: 65-04, Section: B, page: 1842.
500 $a Chair: Sheri J. Lillard.
502 $a Thesis (Ph.D.)--University of California, Riverside, 2004.
520 $a The work presented herein describes the development of capillary electrophoresis (CE)-based methodology for the analysis of nucleic acids originating from individual cells. Three distinct approaches were used to accomplish this goal: (1) evaluation of nucleic acid dyes for DNA detection; (2) the study of DNA and RNA migration in the presence of electroosmotic flow; and (3) the development of a method to measure single-cell messenger RNA (mRNA) using the reverse transcriptase-polymerase chain reaction (RT-PCR).
520 $a Initial studies involved the examination of nucleic acid dyes, in conjunction with laser-induced fluorescence (LIF) detection, for the visualization of DNA restriction fragments and single-cell RNA. Specifically, SBYR Gold and SYBR Green I were used to detect RNA from single cells or a double-stranded (ds)DNA ladder following CE. Both 275- and 488-nm excitation sources were assessed. Spectral characterization of each dye was performed, followed by a detailed comparison of the detection sensitivity for dsDNA and RNA samples.
520 $a In the next phase, a method to monitor DNA and RNA migration in the presence of electroosmotic flow (EOF) was developed. As a consequence of their high negative charge density, the free-solution electrophoretic mobility of nucleic acids opposes EOF. Thus under conditions of moderate EOF, DNA may be selectively ejected from a capillary into an aliquot of water. This phenomenon was initially applied to the co-detection of protein and RNA from cell lysate samples, as EOF retains positively-charged species within the capillary. The ejected RNA was analyzed on a separate CE-LIF system. In later studies, a detailed characterization of this process was performed using DNA restriction fragments and the dsDNA product of the single-cell RT-PCR.
520 $a Finally, an approach was established to evaluate single-locus and multiplex gene expression of individual cells using RT-PCR and CE-LIF. Preliminary studies involved the development of the RT-PCR protocol and examination of cell lysis procedures for the measurement of single-cell beta-actin mRNA. Products were visualized using a hydroxypropylmethylcellulose sieving matrix containing ethidium bromide. This method was subsequently optimized and applied to the simultaneous amplification of beta-actin and estrogen receptor alpha genes, representing the first demonstration of the detection of single-cell multiplex products by CE.
590 $a School code: 0032.
650 4 $a Chemistry, Analytical. $3 586156
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710 2 0 $a University of California, Riverside. $3 791122
773 0 $t Dissertation Abstracts International $g 65-04B.
790 1 0 $a Lillard, Sheri J., $e advisor
790 $a 0032
791 $a Ph.D.
792 $a 2004
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3130287