Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products
First Claim
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1. 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.
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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.
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Citations
29 Claims
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1. In an improved method for sequencing DNA wherein the following steps are conventional:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 28)
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12. A method for ioslating primer extension products from template-directed extension reactions comprising the following steps:
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(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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 29)
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23. A method for isolating primer extension products from template-directed polymerase extension reactions comprising the following steps;
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(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.
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24. A method for isolating primer extension products from template-directed polymerase reactions comprising the following steps:
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(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.
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- 25. 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.
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27. 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.
- C. to 100°
Specification