An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA

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Title: An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA
Author: Chen, Xiaojia
Abstract: Invented in the 1950s , gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs . Conventional gel electrophoresis can successfully separate DNA fragments from several base pairs to a few tens of kilo base pairs , beyond which a point is reached that DNA molecules cannot be resolved due to the size independent mobility . In this case , pulsed field gel electrophoresis (PFGE ) was introduced to extend the range of DNA fragment sizes that can be effectively separated . But despite the incredible success of PFGE techniques , some important drawbacks remain . First , separation time is extremely long , ranging from several hours to a few days . Second , detection methods still rely on staining the gel after the run . Real time observation and study of band migration behavior is impossible due to the large size of the PFGE device . Finally , many commercial PFGE instruments are relatively expensive , a factor that can limit their accessibility both for routine analytical and preparative use as well as for performing fundamental studies . In this research , a miniaturized PFGE device was constructed with dimension 2cm x 2 .6cm , capable of separating DNA fragments ranging from 2 .5kb to 32kb within three hours using low voltage . The separation process can be observed in real time under a fluorescence microscope mounted with a cooled CCD camera . Resolution and mobility of the sample were measured to test the efficiency of the device . We also explored manipulating DNA fragments by end labeling DNA molecules with quantum dot nanocrystals . The quantum dot -DNA conjugates can be further modified through binding interactions with biotinylated single -stranded DNA primers . Single molecule visualization was performed during gel electrophoresis and the extension length , entanglement probability and reorientation time of different conjugates were measured to study their effect on DNA migration through the gel . Finally , electrophoresis of DNA conjugates was performed in the miniaturized PFGE device , and shaper bands were observed compared with the non end -labeled sample . Furthermore , by end -labeling DNA with quantum dots , the migration distance of shorter fragments is reduced , providing the possibility of separating a wider range of DNA fragment sizes on the same gel to achieve further device miniaturization .
URI: http : / /hdl .handle .net /1969 .1 /ETD -TAMU -2898
Date: 2009-05-15

Citation

An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA. Available electronically from http : / /hdl .handle .net /1969 .1 /ETD -TAMU -2898 .

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