Method of gene mapping
First Claim
1. A method for mapping a DNA segment made up of duplex strands of nucleotides bycleaving the segment with a first restriction enzyme to produce fragments of DNA, each having a cleaved end,attaching a reporter specific to a cleaved end nucleotide in each fragment,cleaving the DNA fragments with a second restriction enzyme to produce short fragments,separating the short fragments according to size, andanalyzing the short, separated fragments for the presence of reporters, the size and reporter identity being indicative of the character of each DNA fragment.
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Accused Products
Abstract
The method described characterizes each DNA segment to be mapped by cleaving it to produce DNA fragments which are then end labeled with a reporter(s) specific to the end nucleotides of each fragment. The labeled fragments are again cleaved to produce short fragments which are separated according to size. The short fragments are analyzed as to report identify and size which is indicative of the character of each fragment. By derivatizing the cleaved ends of the primary cleaved fragments, the labeling may be delayed until the second cleavage. Prior to the labeling the derivatized fragments, all underivatized fragments are removed, the derivatized fragments being immobilized.
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Citations
39 Claims
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1. A method for mapping a DNA segment made up of duplex strands of nucleotides by
cleaving the segment with a first restriction enzyme to produce fragments of DNA, each having a cleaved end, attaching a reporter specific to a cleaved end nucleotide in each fragment, cleaving the DNA fragments with a second restriction enzyme to produce short fragments, separating the short fragments according to size, and analyzing the short, separated fragments for the presence of reporters, the size and reporter identity being indicative of the character of each DNA fragment.
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11. A method of mapping DNA segments made up of duplex strands of nucleotides using a reporter comprising:
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reacting a mixture of DNA segments and a first restriction enzyme that cleaves the DNA to provide DNA fragments each with 5'"'"' or 3'"'"' overhang strands and a corresponding 3'"'"' or 5'"'"' recessed strand or with blunt ends, incubating the DNA fragments with a DNA polymerase and a mixture of unlabeled deoxynucleotides and specific reporter labeled dideoxynucleotides to add nucleotides to the recessed strands complementary to the nucleotides in the overhang strands, reacting the incubated DNA fragments with a second restriction enzyme to produce short DNA fragments some of which include the reporter labeled dideoxynucleotides and hence are reporter labeled, separating the short DNA fragments according to size, and detecting a reporter for each labeled fragment thereby identifying the 3'"'"' terminal added dideoxynucleotides. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:
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(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end, (b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair, (c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments, (d) binding the short fragments to the immobilized member, leaving a free end nucleotide on each derivatized fragment, (e) separating the derivatized fragments from the non-derivatized fragments, (f) attaching a reporter to one of the free end nucleotides in each derivatized shorter fragment, (g) separating the reporter labeled, separated fragments from the solid support, and (h) fractionating the reporter labeled fragments according to size. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding substances (one and the other), one substance being immobilized on a solid support, by the steps of:
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cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end, attaching the other substance to the cleaved ends to provide anchor ends, cleaving the DNA fragments with a second restriction enzyme to provide shorter DNA fragments and free ends, separating shorter fragments with an anchor end from the remaining shorter fragments by binding the anchor ends to the one substance, attaching reporter labeled nucleotides to the free end of each separated shorter fragment, removing the separated shorter fragments from the one substance, fractionating the removed shorter fragments.
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38. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:
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(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end, (b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair, (c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments, the second cleaved ends being non-derivatized, (d) attaching a reporter to each of the non-derivatized ends of the short fragments, (e) binding the short fragments with derivatized ends to the one member, (f) separating the bound, derivatized fragments, some of which are reporter-labeled at one end, from the fragments that are not derivatized at either end, (g) separating the bound, reporter-labeled fragments from the solid support, (h) fractionating the reporter labeled fragments according to size.
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39. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:
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(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end, (b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair, (c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments, binding the derivatized fragments to the one member, (d) cleaving the bound DNA fragments with a second restriction enzyme to produce short DNA fragments, some of which have one bound, derivatized and one free end and some of which are not derivatized at either end, (e) separating the bound short fragments from the unbound short fragments, (f) attaching a reporter to one of the free end nucleotides in each bound short fragment, (g) separating the reporter-labeled, bound fragments from the solid support, (h) fractionating the reporter labeled fragments according to size.
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Specification