DNA sequencing using fluorescence background electroblotting membrane
First Claim
1. A method for the multiplex sequencing of DNA comprising:
- (a) providing a target DNA sample to be sequenced wherein the DNA fragments have been chain terminated and separated according to a specific adenine, guanine, cytosine or thymine base groupings;
(b) subjecting base separated groupings of target DNA fragments to gel electrophoresis to resolve the DNA fragments in each base grouping by chain length;
(c) electroblotting said resolved target DNA fragments from said gel onto the surface of a non-aromatic polymeric microporous membrane said membrane being selected from the group consisting of hydrocarbons, fluorocarbons, chlorofluorocarbons, vinyl alcohols and vinyl chlorides and copolymers and blends thereof and exhibiting low background fluorescence said membrane having been surface modified by subjecting said membrane to an aminating agent in the presence of radio frequency plasma discharge or microwave frequency plasma discharge to contain amino groups in order to physically adsorb said DNA fragments on said membrane surface and washing said membrane to remove unadsorbed DNA fragments;
(d) treating said membrane containing said physically adsorbed DNA fragments with crosslinking means to chemically bind said DNA fragments to said membrane through said amino groups contained on the surface thereof;
(e) subjecting said chemically bound DNA fragments on said membrane to hybridization probing with a tagged probe specific to the sequence of the DNA fragments and washing said membranes to remove probe which has not been hybridized; and
(f) detecting and reading said tagged probes hybridized to said target DNA fragments.
2 Assignments
0 Petitions
Accused Products
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 the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.
144 Citations
26 Claims
-
1. A method for the multiplex sequencing of DNA comprising:
-
(a) providing a target DNA sample to be sequenced wherein the DNA fragments have been chain terminated and separated according to a specific adenine, guanine, cytosine or thymine base groupings; (b) subjecting base separated groupings of target DNA fragments to gel electrophoresis to resolve the DNA fragments in each base grouping by chain length; (c) electroblotting said resolved target DNA fragments from said gel onto the surface of a non-aromatic polymeric microporous membrane said membrane being selected from the group consisting of hydrocarbons, fluorocarbons, chlorofluorocarbons, vinyl alcohols and vinyl chlorides and copolymers and blends thereof and exhibiting low background fluorescence said membrane having been surface modified by subjecting said membrane to an aminating agent in the presence of radio frequency plasma discharge or microwave frequency plasma discharge to contain amino groups in order to physically adsorb said DNA fragments on said membrane surface and washing said membrane to remove unadsorbed DNA fragments; (d) treating said membrane containing said physically adsorbed DNA fragments with crosslinking means to chemically bind said DNA fragments to said membrane through said amino groups contained on the surface thereof; (e) subjecting said chemically bound DNA fragments on said membrane to hybridization probing with a tagged probe specific to the sequence of the DNA fragments and washing said membranes to remove probe which has not been hybridized; and (f) detecting and reading said tagged probes hybridized to said target DNA fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
-
- 18. A method of aminating the surface of a non-aromatic polymeric microporous membrane exhibiting low background fluoroescence, wherein said membrane is selected from the group consisting of hydrocarbons, fluorocarbons, chlorofluorocarbons, vinyl alcohols and vinyl chlorides and copolymers and blends thereof, which comprises subjecting said membrane to an aminating agent in the presence of radio frequency plasma discharge or microwave frequency plasma discharge.
Specification