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US5484701A
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Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products
Assignee: EIDP Inc
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Filing: 1992-01-31
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Granted: 1996-01-16
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Expires: 2012-01-31
Abstract
Abstract A simplified method for isolating primer extension products and generating them in a form appropriate for electrophoresis is disclosed. The method is compatible with automated DNA sequencing procedures.
Inventors
- 👤 Anthony J. Cocuzza
- 👤 Frank W. Hobbs, Jr.
- 👤 Robert J. Zagursky
- 👤 Neil A. Straus
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 14:54
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q; C12Q1/00; C12Q1/68; C12Q1/6806; C12Q1/6813; C12Q1/6834; C12Q1/6844; C12Q1/6848; C12Q1/686; C12Q1/6869
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
174 words
In an improved method for sequencing DNA wherein the following steps are conventional: (a) extending a primer by means of a template-directed primer extension reaction; and (b) analyzing the primer extension products by means of gel electrophoresis, the improvement comprising: (i) extending a biotinylated primer by means of a template-directed primer extension reaction to produce biotinylated primer extension products; (ii) complexing the biotin of said biotinylated primer extension products of step (i) with a biotin-binding protein immobilized on a solid support, said complexing performed either before or after separating the template from the biotinylated primer extension products of step (i); (iii) separating physically the complexed biotinylated primer extension products of step (ii) from the liquid phase of the primer extension reaction; (iv) treating the complex of step (iii) with formamide compatible with gel-electrophoresis to dissociate the biotin of said biotinylated primer extension products from the immobilized biotin-binding protein; (v) analyzing the biotinylated primer extension products of step (iv) by means of gel electrophoresis; and (vi) sequencing the DNA of the primer extension products obtained.
Claim 2
Dependent Claim
174 words
In an improved method for sequencing DNA wherein the following steps are conventional: (a) extending a primer by means of a template-directed primer extension reaction; and (b) analyzing the primer extension products by means of gel electrophoresis, the improvement comprising: (i) extending a biotinylated primer by means of a template-directed primer extension reaction to produce biotinylated primer extension products; (ii) complexing the biotin of said biotinylated primer extension products of step (i) with a biotin-binding protein immobilized on a solid support, said complexing performed either before or after separating the template from the biotinylated primer extension products of step (i); (iii) separating physically the complexed biotinylated primer extension products of step (ii) from the liquid phase of the primer extension reaction; (iv) treating the complex of step (iii) with formamide compatible with gel-electrophoresis to dissociate the biotin of said biotinylated primer extension products from the immobilized biotin-binding protein; (v) analyzing the biotinylated primer extension products of step (iv) by means of gel electrophoresis; and (vi) sequencing the DNA of the primer extension products obtained.
Claim 3
Dependent Claim
27 words
The method of claim 1 wherein the biotinylated primer is extended in step (i) by means of a template, a polymerase, and one or more nucleoside triphosphates.
Claim 4
Dependent Claim
21 words
The method of claim 1 wherein the biotin in the biotinylated primer is linked through the 5'-hydroxyl group of the primer.
Claim 5
Dependent Claim
26 words
The method of claim 1 wherein the biotinylated primer extension products of step (i) are treated with a strong base, heat, a single-strand nuclease, or formamide.
Claim 6
Dependent Claim
17 words
The method of claim 4 wherein the products of step (i) are treated with a strong base.
Claim 7
Dependent Claim
23 words
The method of claim 4 wherein the products of step (i) are treated with heat at a temperature between 25° and 100° C.
Claim 8
Dependent Claim
17 words
The method of claim 4 wherein the products of step (i) are treated with a single-strand nuclease.
Claim 9
Dependent Claim
30 words
The method of claim 1 wherein, in step (ii), the solid support is selected from the group consisting of polymeric beads, and magnetic particles, and paper, plastic and glass surfaces.
Claim 10
Dependent Claim
13 words
The method of claim 8 wherein the magnetic particles are chromium dioxide particles.
Claim 11
Dependent Claim
24 words
The method of claim 2, wherein, in step (ii), the biotin-binding protein is selected from the group consisting of avidin, streptavidin, and anti-biotin antibodies.
Claim 12
Dependent Claim
20 words
The method of claim 1 wherein said electrophoresis-compatible formamide is heated to a temperature about 25° C. to 100° C.
Claim 13
Independent Claim
123 words
A method for ioslating primer extension products from template-directed extension reactions comprising the following steps: (a) complexing the biotin of a biotinylated primer to a biotin-binding protein immobilized on a solid support; (b) extending the complexed biotinylated primer of step (a) by means of a template-directed primer extension reaction; (c) separating the template from the complexed biotinylated primer extension products of step (b); (d) separating physically the complexed biotinylated primer extension products of step (c) from the liquid phase of the primer extension reaction; (e) treating the complex of step (d) with formamide to dissociate the biotin of said biotinylated primer extension products from the biotin-binding protein immobilized on a solid support; and (f) sequencing the DNA of the primer extension products obtained.
Claim 14
Independent Claim
123 words
A method for ioslating primer extension products from template-directed extension reactions comprising the following steps: (a) complexing the biotin of a biotinylated primer to a biotin-binding protein immobilized on a solid support; (b) extending the complexed biotinylated primer of step (a) by means of a template-directed primer extension reaction; (c) separating the template from the complexed biotinylated primer extension products of step (b); (d) separating physically the complexed biotinylated primer extension products of step (c) from the liquid phase of the primer extension reaction; (e) treating the complex of step (d) with formamide to dissociate the biotin of said biotinylated primer extension products from the biotin-binding protein immobilized on a solid support; and (f) sequencing the DNA of the primer extension products obtained.
Claim 15
Dependent Claim
17 words
The method of claim 12 wherein the biotin is linked through the 5'-hydroxyl group of the primer.
Claim 16
Dependent Claim
29 words
The method of claim 12 wherein, in step a, the solid support is selected from the group consisting of polymeric beads, magnetic particles, and paper, plastic and glass surfaces.
Claim 17
Dependent Claim
13 words
The method of claim 14 wherein the magnetic particles are chromium dioxide particles.
Claim 18
Dependent Claim
24 words
The method of claim 12 wherein, in step a, the biotin-binding protein is selected from the group consisting of avidin, streptavidin, and anti-biotin antibodies.
Claim 19
Dependent Claim
27 words
The method of claim 12 wherein the biotinylated primer is extended in step b by means of a template, a polymerase, and one or more nucleoside triphosphates.
Claim 20
Dependent Claim
26 words
The method of claim 12 wherein step c is accomplished by treating the products of step b with a strong base, heat, or a single-strand nuclease.
Claim 21
Dependent Claim
17 words
The method of claim 12 wherein the products of step b are treated with a strong base.
Claim 22
Dependent Claim
25 words
The method of claim 12 wherein the products of step b are treated with heat at a temperature of about 25° C. to 100° C.
Claim 23
Dependent Claim
17 words
The method of claim 12 wherein the products of step b are treated with a single-strand nuclease.
Claim 24
Dependent Claim
20 words
The method of claim 12 wherein the electrophoresis-compatible formamide is heated to a temperature about 25° C. to 100° C.
Claim 25
Independent Claim
148 words
A method for isolating primer extension products from template-directed polymerase extension reactions comprising the following steps; (a) extending a 5'-biotinylated primer by means of a template-directed polymerase extension reaction; (b) separating the template from the 5'-biotinylated extension products of step (a) by the use of mung bean nuclease; (c) complexing the biotin of the 5'-biotinylated extension products of step (a) with streptavidin on streptavidin-coated chromium dioxide particles; (d) separating physically the complex of step (c) from the liquid phase of the primer extension reaction by means of a magnet; (e) treating the complex of step (d) with formamide heated to a temperature between about 25° C. to 100° C. to dissociate the biotin of the 5'-biotinylated primer extension products from the streptavidin-coated chromium dioxide particles; and (f) resolving the size the 5'-biotinylated primer extension products of step (e) by means of DNA sequencing of the primer extension products.
Claim 26
Independent Claim
148 words
A method for isolating primer extension products from template-directed polymerase extension reactions comprising the following steps; (a) extending a 5'-biotinylated primer by means of a template-directed polymerase extension reaction; (b) separating the template from the 5'-biotinylated extension products of step (a) by the use of mung bean nuclease; (c) complexing the biotin of the 5'-biotinylated extension products of step (a) with streptavidin on streptavidin-coated chromium dioxide particles; (d) separating physically the complex of step (c) from the liquid phase of the primer extension reaction by means of a magnet; (e) treating the complex of step (d) with formamide heated to a temperature between about 25° C. to 100° C. to dissociate the biotin of the 5'-biotinylated primer extension products from the streptavidin-coated chromium dioxide particles; and (f) resolving the size the 5'-biotinylated primer extension products of step (e) by means of DNA sequencing of the primer extension products.
Claim 27
Independent Claim
140 words
A method for isolating primer extension products from template-directed polymerase reactions comprising the following steps: (a) extending a 5'-biotinylated oligonucleotide primer by means of a template-directed polymerase extension reaction; (b) separating the template from the 5'-biotinylated extension products of step (a) by heating at a temperature of about 25° C. to 100° C.; (c) complexing the biotin of the 5'-biotinylated extension products of step (a) with streptavidin on streptavidin-coated chromium dioxide particles; (d) separating physically the complex of step (c) from the liquid phase of the primer extension reaction by means of a magnet; (e) treating the complex of step (d) with formamide heated to a temperature between about 25° C. to 100° C.; (f) analyzing the 5'-biotinylated primer extension products of step (d) by means of DNA sequencing; and (g) sequencing the DNA of the primer extension products obtained.
Claim 28
Independent Claim
140 words
A method for isolating primer extension products from template-directed polymerase reactions comprising the following steps: (a) extending a 5'-biotinylated oligonucleotide primer by means of a template-directed polymerase extension reaction; (b) separating the template from the 5'-biotinylated extension products of step (a) by heating at a temperature of about 25° C. to 100° C.; (c) complexing the biotin of the 5'-biotinylated extension products of step (a) with streptavidin on streptavidin-coated chromium dioxide particles; (d) separating physically the complex of step (c) from the liquid phase of the primer extension reaction by means of a magnet; (e) treating the complex of step (d) with formamide heated to a temperature between about 25° C. to 100° C.; (f) analyzing the 5'-biotinylated primer extension products of step (d) by means of DNA sequencing; and (g) sequencing the DNA of the primer extension products obtained.
Claim 29
Independent Claim
30 words
A method for dissociating a complex, said complex consisting essentially of biotinylated primer extension products and solid-supported biotin-binding protein immobilized on a solid support, comprising treating said complex with formamide.
Claim 30
Independent Claim
30 words
A method for dissociating a complex, said complex consisting essentially of biotinylated primer extension products and solid-supported biotin-binding protein immobilized on a solid support, comprising treating said complex with formamide.
Claim 31
Dependent Claim
20 words
The method of claim 25 wherein the formamide is heated to a temperature between about 25° C. to 100° C.
Claim 32
Independent Claim
36 words
A method for dissociating a complex, said complex consisting essentially of a biotinylated nucleic acid and a biotin-binding protein, comprising treating said complex with formamide heated to a temperature between about 25° C. to 100° C.
Claim 33
Independent Claim
36 words
A method for dissociating a complex, said complex consisting essentially of a biotinylated nucleic acid and a biotin-binding protein, comprising treating said complex with formamide heated to a temperature between about 25° C. to 100° C.
Claim 34
Dependent Claim
26 words
The method of claim 6, 11, 13, 20, 22, 23,, 24, 25 or 27 wherein the formamide is heated to a temperature of about 95° C.
Claim 35
Dependent Claim
15 words
The method of claim 7 or 21 wherein the single-strand nuclease is mung bean nuclease.
References Cited (Backward Citations)
EP0097373B1
Cited
EP0192168A2
Cited
WO1987003911A1
Cited
WO1987007645A1
Cited
WO1988010313A1
Cited
WO1989009282A1
Cited
WO1989012063A1
Cited
US4908453A
Cited
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