Patent US5217863A successfully refreshed from Google Patents.
US5217863A
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Detection of mutations in nucleic acids
Assignee: Medical Research Council
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Filing: 1991-12-26
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Granted: 1993-06-08
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Expires: 2011-12-26
Abstract
Abstract A method of detecting point mutation in nucleic acids is described which comprises hybridizing a piece of control DNA or RNA without mutations with a piece of test DNA or RNA generally corresponding to the test DNA or RNA but possibly with mutations to produce a heteroduplex, treating the heteroduplex with hydroxylamine or osmium tetroxide and with piperidine, and subjecting the resulting material to separation treatment. Individual strands of both senses of control nucleic acid can be labelled in turn allowing detection of all possible mutations.
Inventors
- 👤 Richard G. H. Cotton
- 👤 Robert D. Campbell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 13:23
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6883; C12Q1/6813; C12Q1/6827; C12Q1/70; C12Q1/701; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
101 words
A method for detecting point mutations in a nucleic acid which comprises hybridizing a piece of control DNA or RNA that contains no mutations with a piece of test DNA or RNA that contains mutations to produce a linear heteroduplex; treating said linear heteroduplex with a solution containing an effective amount of aqueous hydroxylamine, or a solution containing an effective amount of aqueous osmium tetroxide and then with piperidine thereby causing cleavage at mismatched thymine or cytosine, subjecting the cleaved DNA or RNA to a separation treatment, and detecting fragments of the cleaved DNA or RNA as indicative of point mutations.
Claim 2
Independent Claim
101 words
A method for detecting point mutations in a nucleic acid which comprises hybridizing a piece of control DNA or RNA that contains no mutations with a piece of test DNA or RNA that contains mutations to produce a linear heteroduplex; treating said linear heteroduplex with a solution containing an effective amount of aqueous hydroxylamine, or a solution containing an effective amount of aqueous osmium tetroxide and then with piperidine thereby causing cleavage at mismatched thymine or cytosine, subjecting the cleaved DNA or RNA to a separation treatment, and detecting fragments of the cleaved DNA or RNA as indicative of point mutations.
Claim 3
Dependent Claim
28 words
A method according to claim 1, wherein said heteroduplex is incubated with from 2 to 2.5M hydroxylamine hydrochloride for up to 2 hours at 37° C. at pH6.
Claim 4
Dependent Claim
27 words
A method according to claim 1, wherein said heteroduplex is incubated with 2.4% w/v osmium tetroxide for up to 1 hour at 37° C. at pH 7.7.
Claim 5
Dependent Claim
11 words
A method according to claim 1, wherein separation is by electrophoresis.
Claim 6
Dependent Claim
22 words
A method according to claim 1, wherein either strand of said control or test DNA or RNA is end-labelled or internally labelled.
Claim 7
Dependent Claim
20 words
A method according to claim 5, wherein only one strand in the heteroduplex is labelled, enabling indirect detection of mutations.
Claim 8
Dependent Claim
20 words
A method according to claim 5, wherein said label is a radioactive label, a fluorescent label or an enzyme label.
Claim 9
Dependent Claim
23 words
A method according to claim 1, wherein said treatment with hydroxylamine or osmium tetroxide is carried out on separate samples of said heteroduplex.
Claim 10
Dependent Claim
37 words
A method according to claim 1, wherein said test nucleic acid is RNA which is reacted to produce complementary test DNA and then said complementary test DNA is hybridized with complementary control DNA to produce said heteroduplex.
Claim 11
Independent Claim
134 words
A method for detecting all point mutations in a nucleic acid which comprises: a) hybridizing a piece of labeled control DNA or RNA that contains no mutations with a piece of test DNA or RNA that contains mutations to produce a linear heteroduplex; b) hybridizing a piece complementary to said control DNA or RNA with a piece complementary to said test DNA or RNA; c) dividing each sample in two, one portion being treated with a solution containing an effective amount of aqueous osmium tetroxide and the other with a solution containing aqueous hydroxylamine then treating all four sample portions with piperidine, thereby causing cleavage at mismatched thymine or cytosine; d) subjecting the resulting material to a separation treatment; and e) detecting the fragments of cleaved DNA or RNA as indicative of point mutations.
Claim 12
Independent Claim
134 words
A method for detecting all point mutations in a nucleic acid which comprises: a) hybridizing a piece of labeled control DNA or RNA that contains no mutations with a piece of test DNA or RNA that contains mutations to produce a linear heteroduplex; b) hybridizing a piece complementary to said control DNA or RNA with a piece complementary to said test DNA or RNA; c) dividing each sample in two, one portion being treated with a solution containing an effective amount of aqueous osmium tetroxide and the other with a solution containing aqueous hydroxylamine then treating all four sample portions with piperidine, thereby causing cleavage at mismatched thymine or cytosine; d) subjecting the resulting material to a separation treatment; and e) detecting the fragments of cleaved DNA or RNA as indicative of point mutations.
Claim 13
Dependent Claim
33 words
A method according to claim 10, wherein said other portions of said samples are each incubated with from 2 to 2.5M hydroxylamine hydrochloride for up to 2 hours at 37° C. at pH6.
Claim 14
Dependent Claim
32 words
A method according to claim 10, wherein said one portions of said samples are each incubated with 2.4% w/v osmium tetroxide for up to 1 hour at 37° C. at pH 7.7.
Claim 15
Dependent Claim
12 words
A method according to claim 10, wherein said separation treatment is electrophoresis.
Claim 16
Dependent Claim
25 words
A method according to claim 10, wherein said control DNA or RNA complementary to said test DNA or RNA is end labelled or internally labelled.
Claim 17
Dependent Claim
19 words
A method according to claim 14, wherein said label is a radioactive label, a fluorescent label or enzyme label.
References Cited (Backward Citations)
GB2179735A
Cited
US4794075A
Cited
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