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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 amino groups contained on the surface. 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 the 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 amountmore » of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings« less

Inventors:
; ;
Issue Date:
OSTI Identifier:
7272788
Patent Number(s):
5112736 A
Application Number:
PPN: US 7-365693
Assignee:
Univ. of Utah, Salt Lake City, UT (United States); Brigham Young Univ., Provo, UT (United States) PTO; EDB-94-084023
DOE Contract Number:  
FG02-88ER60700
Resource Type:
Patent
Resource Relation:
Patent File Date: 14 Jun 1989
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA SEQUENCING; MEMBRANES; FLUORESCENCE; CHEMICAL BONDS; CROSS-LINKING; DNA HYBRIDIZATION; CHEMICAL REACTIONS; HYBRIDIZATION; LUMINESCENCE; POLYMERIZATION; STRUCTURAL CHEMICAL ANALYSIS; 550200* - Biochemistry

Citation Formats

Caldwell, K.D., Chu, T.J., and Pitt, W.G. DNA sequencing using fluorescence background electroblotting membrane. United States: N. p., 1992. Web.
Caldwell, K.D., Chu, T.J., & Pitt, W.G. DNA sequencing using fluorescence background electroblotting membrane. United States.
Caldwell, K.D., Chu, T.J., and Pitt, W.G. Tue . "DNA sequencing using fluorescence background electroblotting membrane". United States.
@article{osti_7272788,
title = {DNA sequencing using fluorescence background electroblotting membrane},
author = {Caldwell, K.D. and Chu, T.J. and Pitt, W.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 amino groups contained on the surface. 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 the 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 membranes may be reprobed numerous times. No Drawings},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1992},
month = {5}
}