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Title: DNA sequencing using fluorescence background electroblotting membrane

Abstract

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when themore » available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.« less

Inventors:
 [1];  [1];  [2]
  1. Salt Lake City, UT
  2. Orem, UT
Issue Date:
Research Org.:
Univ. of Utah, Salt Lake City, UT (United States)
OSTI Identifier:
868284
Patent Number(s):
5112736
Assignee:
University of Utah (Salt Lake City, UT); Brigham Young University (Provo, UT)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10T - TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
DOE Contract Number:  
FG02-88ER60700
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
dna; sequencing; fluorescence; background; electroblotting; membrane; method; multiplex; disclosed; comprises; specific; base; terminated; fragments; resolved; electrophoresis; surface; neutral; non-aromatic; polymeric; microporous; exhibiting; modified; contain; amino; polypropylene; membranes; preferably; introduction; accomplished; subjecting; radio; microwave; frequency; plasma; discharge; presence; aminating; agent; ammonia; containing; physically; adsorbed; treated; crosslinking; means; radiation; glutaraldehyde; spray; chemically; bind; smino; contained; bound; subjected; hybridization; probing; tagged; probe; sequence; tagging; fluorophores; radioisotopes; probes; hybridized; target; detected; laser; induced; detection; autoradiograms; aminated; fluorescent; allows; reading; available; amount; sequenced; membrances; reprobed; numerous; times; fluorescence detection; microwave frequency; plasma discharge; porous membrane; dna fragments; dna sequencing; laser induced; target dna; dna fragment; multiplex sequencing; surface modified; chemically bound; microporous membrane; induced fluorescence; specific base; chemically bind; wave frequency; fluorescent detection; frequency plasma; /435/436/

Citation Formats

Caldwell, Karin D, Chu, Tun-Jen, and Pitt, William G. DNA sequencing using fluorescence background electroblotting membrane. United States: N. p., 1992. Web.
Caldwell, Karin D, Chu, Tun-Jen, & Pitt, William G. DNA sequencing using fluorescence background electroblotting membrane. United States.
Caldwell, Karin D, Chu, Tun-Jen, and Pitt, William G. Wed . "DNA sequencing using fluorescence background electroblotting membrane". United States. https://www.osti.gov/servlets/purl/868284.
@article{osti_868284,
title = {DNA sequencing using fluorescence background electroblotting membrane},
author = {Caldwell, Karin D and Chu, Tun-Jen and Pitt, William G},
abstractNote = {A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1992},
month = {1}
}

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