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Title: High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

Abstract

Sample preparation has been one of the major bottlenecks for many high throughput analyses. 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 homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author 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. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. 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 anmore » accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection 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 mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an attractive alternative to current methods for genetic analysis and disease diagnosis.« less

Authors:
 [1]
  1. Iowa State Univ., Ames, IA (United States)
Publication Date:
Research Org.:
Ames Lab., Ames, IA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
804158
Report Number(s):
IS-T 2002
TRN: US200302%%572
DOE Contract Number:  
W-7405-Eng-82
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: TH: Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); PBD: 27 May 2002
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; BLOOD; CAPILLARIES; CATALYSIS; DNA; DNA SEQUENCING; ELECTROPHORESIS; ENZYMES; FLUORESCENCE; GENETICS; HOMOGENEOUS CATALYSIS; PLASMIDS; SAMPLE PREPARATION

Citation Formats

Zhang, Yonghua. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique. United States: N. p., 2000. Web. doi:10.2172/804158.
Zhang, Yonghua. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique. United States. doi:10.2172/804158.
Zhang, Yonghua. Sat . "High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique". United States. doi:10.2172/804158. https://www.osti.gov/servlets/purl/804158.
@article{osti_804158,
title = {High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique},
author = {Zhang, Yonghua},
abstractNote = {Sample preparation has been one of the major bottlenecks for many high throughput analyses. 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 homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author 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. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. 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, they demonstrated that capillary electrophoresis with UV detection 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 mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an attractive alternative to current methods for genetic analysis and disease diagnosis.},
doi = {10.2172/804158},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2000},
month = {1}
}

Thesis/Dissertation:
Other availability
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