US2020299769A1PendingUtilityA1

Hybridization compositions and methods

66
Assignee: AGILENT TECHNOLOGIES INCPriority: Oct 21, 2011Filed: Apr 7, 2020Published: Sep 24, 2020
Est. expiryOct 21, 2031(~5.2 yrs left)· nominal 20-yr term from priority
Inventors:Steen Hauge Matthiesen
C12Q 1/6832C12Q 1/6841C12Q 1/6881
66
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Claims

Abstract

The invention provides methods and compositions for hybridizing at least one molecule to a target. The invention may, for example, utilize a of cyclic and/or non-cyclic solvent that is non-toxic and may eliminate or reduce the amount of formamide in the hybridization composition.

Claims

exact text as granted — not AI-modified
1 - 26 . (canceled) 
     
     
         27 . A method of hybridizing nucleic acid sequences comprising:
 providing a first nucleic acid sequence,   providing a second nucleic acid sequence,   providing a hybridization composition comprising at least one solvent in an amount effective to denature double-stranded nucleotide sequences, and   combining the first and the second nucleic acid sequence and the hybridization composition for at least a time period sufficient to hybridize the first and second nucleic acid sequences,   wherein the solvent is chosen from 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone, N,N-dimethyl-acetamide and isobutyramide.   
     
     
         28 . A method of hybridizing nucleic acid sequences comprising:
 providing a first nucleic acid sequence in an in situ biological sample, and   applying a hybridization composition comprising a second nucleic acid sequence and at least one solvent in an amount effective to denature double-stranded nucleotide sequences to said first nucleic acid sequence for at least a time period sufficient to hybridize the first and second nucleic acid sequences,   wherein the solvent is chosen from 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone, N ,N-dimethyl-acetamide and isobutyramide.   
     
     
         29 - 32 . (canceled) 
     
     
         33 . The method according to  claim 27 , wherein a sufficient amount of energy to hybridize the first and second nucleic acids is provided. 
     
     
         34 . The method according to  claim 33 , wherein the energy is provided by heating the hybridization composition and nucleic acid sequence. 
     
     
         35 . The method according to  claim 33 , wherein the heating step is performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating or heat lamps. 
     
     
         36 . The method according to  claim 27 , wherein the first nucleic acid sequence is double stranded and the second nucleic acid is single stranded. 
     
     
         37 . The method according to  claim 27 , wherein the denaturation and hybridization steps occur separately. 
     
     
         38 . The method according to  claim 27 , wherein the step of hybridizing includes the steps of heating and cooling the hybridization composition and nucleic acid sequences. 
     
     
         39 . The method according to  claim 27 , wherein the step of hybridization takes less than 2 hours. 
     
     
         40 . The method according to  claim 39 , wherein the step of hybridization takes less than 1 hour. 
     
     
         41 . The method according to  claim 40 , wherein the step of hybridization takes less than 30 minutes. 
     
     
         42 . The method according to  claim 41 , wherein the step of hybridization takes less than 15 minutes. 
     
     
         43 . The method according to  claim 42 , wherein the step of hybridization takes less than 5 minutes. 
     
     
         44 . The method according to  claim 27 , wherein the cooling step takes less than 1 hour. 
     
     
         45 . The method according to  claim 44 , wherein the cooling step takes less than 30 minutes. 
     
     
         46 . The method according to  claim 45 , wherein the cooling step takes less than 15 minutes. 
     
     
         47 . The method according to  claim 46 , wherein the cooling step takes less than 5 minutes. 
     
     
         48 . The method according to  claim 27 , wherein the denaturation step is performed at 67° C. 
     
     
         49 . The method according to  claim 27 , wherein the first nucleic acid sequence is in a biological sample. 
     
     
         50 . The method according to  claim 49 , wherein the biological sample is a cytology or histology sample. 
     
     
         51 - 57 . (canceled)

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