Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
231 words
A method for determining whether the DNA contained in a test sample includes a particular nucleic acid sequence, characterized in that it comprises the steps of: a) extracting nucleic acids from the test sample, b) digesting the extracted nucleic acids with restriction enzyme thereby to cleave the DNA or not at a particular sequence, depending on whether or not a restriction enzyme recognition site is present in the sequence, c) treating the product of (b) to form single-stranded nucleic acids, d) contacting the single-stranded nucleic acids produced in (c) with first and second polynucleotide probes which are complementary to respective first and second portions of said sequence to be detected, the two portions being non-overlapping and immediately adjacent to the restriction site in question, such contact being performed under conditions favorable to hybridization of said first and second probes to said sequence to be detected, hybridization with both of said probes being dependent upon whether in step (b) restriction did not occur, said first probe being incorporated with a distinguishable label, e) separating, by means of said second probe, (i) any resulting dual hybridization product comprising said sequence to be detected hybridized to both said labeled first probe and said second probe, from (ii) any unhybridized and single hybridized labeled first probe, and f) by means of said label detecting any of said separated dual hybridization product which may be present.
Claim 2
Independent Claim
231 words
A method for determining whether the DNA contained in a test sample includes a particular nucleic acid sequence, characterized in that it comprises the steps of: a) extracting nucleic acids from the test sample, b) digesting the extracted nucleic acids with restriction enzyme thereby to cleave the DNA or not at a particular sequence, depending on whether or not a restriction enzyme recognition site is present in the sequence, c) treating the product of (b) to form single-stranded nucleic acids, d) contacting the single-stranded nucleic acids produced in (c) with first and second polynucleotide probes which are complementary to respective first and second portions of said sequence to be detected, the two portions being non-overlapping and immediately adjacent to the restriction site in question, such contact being performed under conditions favorable to hybridization of said first and second probes to said sequence to be detected, hybridization with both of said probes being dependent upon whether in step (b) restriction did not occur, said first probe being incorporated with a distinguishable label, e) separating, by means of said second probe, (i) any resulting dual hybridization product comprising said sequence to be detected hybridized to both said labeled first probe and said second probe, from (ii) any unhybridized and single hybridized labeled first probe, and f) by means of said label detecting any of said separated dual hybridization product which may be present.
Claim 3
Dependent Claim
41 words
A method according to claim 1, characterized in that the second probe is in a form which enables the hybridization product of the first probe and said sequence to be detected to be separated from unhybridized and singly hybridized first probe.
Claim 4
Dependent Claim
18 words
A method according to claim 2, characterized in that the second probe is fixed to a solid support.
Claim 5
Independent Claim
28 words
A method according to any of claims 1 to 3, characterized in that each of the probes is independently produced by sub-cloning in a different host vector system.
Claim 6
Independent Claim
80 words
A method according to any of claims 1 to 4, characterized in that the first and second probes are obtained by digestion of a cloned nucleic acid containing material identical with that of the test sample, digestion . producing two nucleic acid fragments, separating the two fragments so that one becomes the second probe, and applying a distinguishable label to that fragment which becomes the first probe, application of the label being effected either before or after digestion or separation.
Claim 7
Dependent Claim
16 words
A method according to claim 5, characterized in that the digestion is effected with restriction enzymes.
Claim 8
Dependent Claim
40 words
A method according to claim 5 or 6, characterized in that the first and second probes are produced by digestion of a hemoglobin gene, the test sample being the hemoglobin gene of a patient being tested for sickle cell anemia.
Claim 9
Dependent Claim
36 words
A method according to claim 7, characterized in that the enzymatic digestion of the test sample is such that it will leave sickle cell anemia r.jcleic acid of a length which can hybridize with both probes.
Claim 10
Dependent Claim
41 words
A method according to claim 8, characterized in that the first and second probes are produced by digestion of a normal hemoglobin gene, hybridization of the digested test sample to both indicating the presence therein of sickle cell anemia nucleic acid.
Claim 11
Dependent Claim
125 words
A kit for determining whether the nucleic acid contained in a test sample includes a particular nucleic acid sequence, comprising (1) first and second probes each including nucleic acid sequences present in the nucleic acid of a test sample, the first probe carrying a distinguishable label and being soluble in a liquid in which the determination will be run, the second probe being fixed on a solid support, and (2) a restriction enzyme which will cleave the test sample or fail to depending on the presence or absence of a particular nucleic acid sequence, whereby a positive determination test sample is capable of hybridizing with both the first and second probes so as thereby to affix the label to the solid support for subsequent reading.
Claim 12
Dependent Claim
125 words
A kit for determining whether the nucleic acid contained in a test sample includes a particular nucleic acid sequence, comprising (1) first and second probes each including nucleic acid sequences present in the nucleic acid of a test sample, the first probe carrying a distinguishable label and being soluble in a liquid in which the determination will be run, the second probe being fixed on a solid support, and (2) a restriction enzyme which will cleave the test sample or fail to depending on the presence or absence of a particular nucleic acid sequence, whereby a positive determination test sample is capable of hybridizing with both the first and second probes so as thereby to affix the label to the solid support for subsequent reading.