US5015569A
Expired / Lapsed
US
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Acceleration of nucleic acid hybridization
Assignee: Leland Stanford Junior University
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Filing: 1989-12-01
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Granted: 1991-05-14
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Expires: 2009-12-01
Abstract
Abstract This invention relates to an improvement in nucleic acid hybridization technology. Nucleic acids bind to complementary partners in a predictable manner such that the detection of complementary partners is possible. The acceleration of the binding process is desired objective and will find broad application in a variety of industrial, medical, and research uses. In particular this invention relates to the acceleration of nucleic acid hybridization by heterogeneous nuclear ribonucleoproteins [hnRNPs].
Inventors
- 👤 Brian W. Pontius
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 20:39
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5306; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; Y; Y10; Y10S; Y10S435/00; Y10S435/82
Claims Analysis
Total Claims: 31
Claim 1
Independent Claim
109 words
A method for accelerating the rate of hybridization between two complementary nucleic acid sequences in an in vitro nucleic acid hybridization assay comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization where the reaction mixture comprises heterogeneous nuclear ribonucleoprotein having a carboxy terminus capable of hnRNP/hnRNP interaction, said ribonucleoprotein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein; and (b) detecting the hybridization of the two complementary nucleic acid sequences with the proviso that neither of the nucleic acid sequences being detected is a polyribouridylic acid.
Claim 2
Independent Claim
109 words
A method for accelerating the rate of hybridization between two complementary nucleic acid sequences in an in vitro nucleic acid hybridization assay comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization where the reaction mixture comprises heterogeneous nuclear ribonucleoprotein having a carboxy terminus capable of hnRNP/hnRNP interaction, said ribonucleoprotein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein; and (b) detecting the hybridization of the two complementary nucleic acid sequences with the proviso that neither of the nucleic acid sequences being detected is a polyribouridylic acid.
Claim 3
Dependent Claim
13 words
A method of claim 1 wherein the ribonucleoprotein is an A1 core protein.
Claim 4
Dependent Claim
14 words
A method of claim 1 wherein the ribonucleoprotein is a mammalian A1 core protein.
Claim 5
Dependent Claim
15 words
A method of claim 1 wherein the carboxy terminus of the ribonucleoprotein is glycine rich.
Claim 6
Dependent Claim
15 words
A method of claim 1 wherein the A1 core protein is derived from a rat.
Claim 7
Dependent Claim
23 words
The method of claim 1 wherein the method further comprises the immobilization of one of the two nucleic acids to a solid support.
Claim 8
Dependent Claim
22 words
The method of claim 1 wherein the method further comprises the labeling of one of the two nucleic acids with a reporter.
Claim 9
Dependent Claim
60 words
A method of claim 1 wherein the method further comprises in step (a) an immobilized nucleic acid, a target nucleic acid complementary to the immobilized nucleic acid and a nucleic acid sequence labeled with a reporter and having a sequence complementary to a portion of the target nucleic acid different from the sequence complementary to the immobilized capture nucleic acid.
Claim 10
Dependent Claim
32 words
A method of claim 1 wherein the amount of heterogeneous ribonucleoprotein is at least about a 5 fold excess by weight of the total amount of nucleic acid in the reaction mixture.
Claim 11
Dependent Claim
32 words
A method of claim 1 wherein the amount of heterogeneous ribonucleoprotein is at least about a 10 fold excess by weight of the total amount of nucleic acid in the reaction mixture.
Claim 12
Dependent Claim
32 words
A method of claim 1 wherein the amount of heterogeneous ribonucleoprotein is at least about a 20 fold excess by weight of the total amount of nucleic acid in the reaction mixture.
Claim 13
Dependent Claim
17 words
A method of claim 1 wherein the nucleic acid sequences are polynucleotides of at least 33 nucleotides.
Claim 14
Dependent Claim
14 words
A method of claim 1 wherein the nucleic acid is a polynucleotide of DNA.
Claim 15
Dependent Claim
14 words
A method of claim 1 wherein the nucleic acid is a polynucleotide of RNA.
Claim 16
Independent Claim
93 words
A method for accelerating the rate of hybridization between two complementary nucleic acid sequences in an in vitro nucleic acid hybridization assay comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization where the reaction mixture comprises heterogeneous nuclear ribonucleoprotein having a carboxy terminus capable of hnRNP/hnRNP interaction, said ribonucleoprotein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein; and (b) detecting the hybridization of the two complementary nucleic acid sequences.
Claim 17
Independent Claim
93 words
A method for accelerating the rate of hybridization between two complementary nucleic acid sequences in an in vitro nucleic acid hybridization assay comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization where the reaction mixture comprises heterogeneous nuclear ribonucleoprotein having a carboxy terminus capable of hnRNP/hnRNP interaction, said ribonucleoprotein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein; and (b) detecting the hybridization of the two complementary nucleic acid sequences.
Claim 18
Dependent Claim
20 words
A method of claim 15 wherein the nucleic acid sequences are a polynucleotides of at least 33 nucleotides in length.
Claim 19
Dependent Claim
14 words
A method of claim 15 wherein the nucleic acid is a polynucleotide of DNA.
Claim 20
Dependent Claim
14 words
A method of claim 15 wherein the nucleic acid is a polynucleotide of RNA.
Claim 21
Dependent Claim
33 words
A method of claim 15 wherein the amount of heterogeneous ribonucleoprotein is at least about a 5 fold excess by weight of the total amount of nucleic acid present in the reaction mixture.
Claim 22
Independent Claim
115 words
A method for conducting a high temperature protein-mediated nucleic acid hybridization assay having a target nucleic acid and a probe nucleic acid comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization, including temperatures at about 45° C. or above, where the reaction mixture comprises a single stranded nucleic acid binding protein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein and where the numbers of probe nucleic acid to target nucleic acid does not approximate a 1 to 1 ratio; and (b) detecting the hybridization of the two complementary nucleic acid sequences.
Claim 23
Independent Claim
115 words
A method for conducting a high temperature protein-mediated nucleic acid hybridization assay having a target nucleic acid and a probe nucleic acid comprising: (a) hybridizing two complementary nucleic acid sequences in a hybridization reaction mixture under conditions permitting nucleic acid hybridization, including temperatures at about 45° C. or above, where the reaction mixture comprises a single stranded nucleic acid binding protein present in an amount sufficient to substantially accelerate the rate of hybridization above the hybridization rate in the absence of the ribonucleoprotein and where the numbers of probe nucleic acid to target nucleic acid does not approximate a 1 to 1 ratio; and (b) detecting the hybridization of the two complementary nucleic acid sequences.
Claim 24
Dependent Claim
26 words
A method of claim 20 where the single stranded nucleic acid binding protein is a heterogeneous nuclear ribonucleoprotein having a carboxy terminus capable of hnRNP/hnRNP interaction.
Claim 25
Dependent Claim
12 words
A method of claim 20 where the temperature is about 65° C.
Claim 26
Dependent Claim
12 words
A method of claim 22 where the nucleic acid sequences are DNA.
Claim 27
Dependent Claim
50 words
A kit for the detection of nucleic acid hybridization comprising a compartment with nucleic acid, a compartment with hybridization reagents, and a compartment with heterogeneous nuclear ribonucleoprotein having a glycine-rich carboxy terminus said ribonucleoprotein with the proviso that neither of the nucleic acid sequences being detected is a polyribouridylic acid.
Claim 28
Dependent Claim
50 words
A kit for the detection of nucleic acid hybridization comprising a compartment with nucleic acid, a compartment with hybridization reagents, and a compartment with heterogeneous nuclear ribonucleoprotein having a glycine-rich carboxy terminus said ribonucleoprotein with the proviso that neither of the nucleic acid sequences being detected is a polyribouridylic acid.
Claim 29
Dependent Claim
13 words
A kit of claim 24 wherein the ribonucleoprotein is an A1 core protein.
Claim 30
Dependent Claim
17 words
A kit of claim 24 further comprising a compartment having a nucleic acid labelled with a reporter.
Claim 31
Dependent Claim
19 words
A kit of claim 26 wherein the reporter is selected from the group consisting of an enzyme and fluorophore.
References Cited (Backward Citations)
No citation records indexed.
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