High throughput sample preparation and analysis for DNA sequencing, PCR and combinatorial screening of catalysis based on capillary array technique

Date
2000-01-01
Authors
Zhang, Yonghua
Major Professor
Advisor
Edward S. Yeung
Committee Member
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Altmetrics
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Research Projects
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Chemistry
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Chemistry
Abstract

The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogenous catalysis based on multiplexed capillary electrophoresis;We first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. Protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation;For PCR based DNA analysis, we demonstrated that UV absorption capillary electrophoresis can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixture was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis based simply on multiplexed UV imaging absorption detection to increase the throughput;We further developed a new methodology-nonaqueous capillary array electrophoresis coupled with microreaction, to address the throughput needs of combinatorial approaches to homogeneous catalysis screening and reaction optimization. Samples were injected directly from reaction vial without dilution and reaction quenching. Buffer compatibility was also found important for reliable 96-capillary array injection. By choosing deferent Pd and base, a combination of 88 different reaction conditions was quickly tested. The analysis time was less than one minute for one sample.

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