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Title: Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis

Abstract

The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time and shortening separation times to approximate real time monitoring. The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 timesmore » faster than the use of a constant field program. The separation of ΦX174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. The relative migration rates of the fragments do not change with temperature but are affected by the applied field. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20°C, all fragments are separated in less than 4 minutes with an average plate number of 2.5 million per meter.« less

Authors:
 [1]
  1. Iowa State Univ., Ames, IA (United States)
Publication Date:
Research Org.:
Ames Lab., IA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
10116369
Report Number(s):
IS-T-1658
ON: DE94005555; TRN: 94:001251
DOE Contract Number:  
W-7405-ENG-82
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: TH: Thesis; PBD: Jul 1993
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; DNA; ELECTROPHORESIS; OPTIMIZATION; CAPILLARY FLOW; GENETIC MAPPING; TIME DEPENDENCE; SENSITIVITY; TWO-DIMENSIONAL ELECTROPHORESIS; 400105; SEPARATION PROCEDURES

Citation Formats

McGregor, David A. Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis. United States: N. p., 1993. Web. doi:10.2172/10116369.
McGregor, David A. Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis. United States. https://doi.org/10.2172/10116369
McGregor, David A. Thu . "Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis". United States. https://doi.org/10.2172/10116369. https://www.osti.gov/servlets/purl/10116369.
@article{osti_10116369,
title = {Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis},
author = {McGregor, David A.},
abstractNote = {The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time and shortening separation times to approximate real time monitoring. The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 times faster than the use of a constant field program. The separation of ΦX174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. The relative migration rates of the fragments do not change with temperature but are affected by the applied field. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20°C, all fragments are separated in less than 4 minutes with an average plate number of 2.5 million per meter.},
doi = {10.2172/10116369},
url = {https://www.osti.gov/biblio/10116369}, journal = {},
number = ,
volume = ,
place = {United States},
year = {1993},
month = {7}
}

Thesis/Dissertation:
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