Hybridization compositions and methods using formamide

Abstract

The invention provides methods and compositions for hybridizing at least one molecule to a target. The composition comprises at least one nucleic acid sequence, formamide, and a hybridization solution, wherein the concentration of formamide is less than or equal to 25%.

1 . A hybridization composition comprising at least one nucleic acid sequence, formamide, and a hybridization solution, wherein the concentration of formamide is less than or equal to 25%. 
     
     
         2 . The hybridization composition according to  claim 1 , wherein the concentration of formamide is less than or equal to 20%. 
     
     
         3 . The hybridization composition according to  claim 1  or  2 , wherein the concentration of formamide is 10% to 15%. 
     
     
         4 . The hybridization composition according to  claim 1  or  2 , wherein the concentration of formamide is 10% to 20%. 
     
     
         5 . The hybridization composition according to  claim 1 ,  2 , or  3 , wherein the concentration of formamide is 15% to 20%. 
     
     
         6 . The hybridization composition according to  claim 1 , wherein the concentration of formamide is 20% to 25%. 
     
     
         7 . The hybridization composition according to  claim 1 , wherein the concentration of formamide is 25%. 
     
     
         8 . The hybridization composition according to any one of the  claims 1  to  5 , wherein the concentration of formamide is 15%. 
     
     
         9 . The hybridization composition according to any one of the  claims 1  to  8 , further comprising at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, blocking agents and combinations thereof. 
     
     
         10 . The hybridization composition according to  claim 9 , wherein the at least one additional component is a combination of a accelerating agent and salts, wherein the accelerating agent is dextran sulfate and the salts are NaCl and/or citrate buffer. 
     
     
         11 . The hybridization composition according to  claim 10 , wherein the dextran sulfate is present at a concentration of 10% to 40%, the NaCl is present at a concentration of 300 mM to 1200 mM, and/or the citrate buffer is present at a concentration of 0 mM to 500 mM. 
     
     
         12 . The hybridization composition according to  claim 11 , wherein the dextran sulfate is present at a concentration of 15% to 30%, the NaCl is present at a concentration of 300 mM to 900 mM, and/or the citrate buffer is present at a concentration of 5 mM to 50 mM. 
     
     
         13 . The hybridization composition according to  claim 9 , wherein the at least one additional component is a combination of a accelerating agent and a buffering agent wherein the accelerating agent is selected from the group consisting of: glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, 1,3 propanediol and combinations thereof, and the buffering agent is citrate buffer. 
     
     
         14 . The hybridization composition according to  claim 13 , wherein the accelerating agent is present at a concentration of 0.1% to 10%, and the citrate buffer is present at a concentration of 1 mM to 50 mM. 
     
     
         15 . The hybridization buffer according to any one of the  claims 10  to  14 , wherein the citrate buffer is present at a concentration of 5 mM to 20 mM. 
     
     
         16 . The hybridization buffer according to any one of the  claims 10  to  15 , wherein the citrate buffer is present at a concentration of 5 mM to 15 mM. 
     
     
         17 . The hybridization buffer according to any one of the  claims 10  to  14 , wherein the citrate buffer is present at a concentration of 10 mM to 50 mM. 
     
     
         18 . The hybridization buffer according to any one of the  claim 10  to  15 , or  17 , wherein the citrate buffer is present at a concentration of 10 mM to 20 mM. 
     
     
         19 . The hybridization buffer according to any one of the  claims 10  to  18 , wherein the citrate buffer is present at a concentration of 10 mM to 15 mM. 
     
     
         20 . The hybridization composition according to any one of the  claims 10  to  12 , wherein the NaCl is present at a concentration of 300 mM to 700 mM. 
     
     
         21 . The hybridization composition according to any one of the  claim 10  to  12 , or  20 , wherein the NaCl is present at a concentration of 400 to 700 mM. 
     
     
         22 . The hybridization composition according to any one of the  claim 10  to  12 ,  20 , or  21 , wherein the NaCl is present at a concentration of 500 to 700 mM. 
     
     
         23 . The hybridization composition according to  claim 9 , wherein the at least one additional component is a blocking agent selected from the group consisting of: total human DNA, COT1 DNA, blocking PNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA. 
     
     
         24 . The hybridization composition according to  claim 23 , wherein the total human DNA, COT1 DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA are present at a concentration of 0.01 to 10 μg/μL. 
     
     
         25 . The hybridization composition according to any one of  claims 1  to  6  or  8  to  24 , comprising 15% formamide, 20% dextran sulfate, 600 mM NaCl, and 10 mM citrate buffer. 
     
     
         26 . The hybridization composition according to any one of  claims 1  to  6  or  8  to  25 , comprising 15% formamide, 20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer, and 0.1 μg/μL COT1 DNA. 
     
     
         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 formamide, 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 concentration of formamide is less than or equal to 25%. 
     
     
         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 formamide to said first nucleic acid sequence for at least a time period sufficient to hybridize the first and second nucleic acid sequences,   
       wherein the formamide is present in a concentration less than or equal to 25%. 
     
     
         29 . A method of hybridizing nucleic acid sequences comprising:
 providing a first nucleic acid sequence,   providing a second nucleic acid sequence,   providing a hybridization composition according to any of  claims 1  to  26 , 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.   
     
     
         30 . A method of hybridizing nucleic acid sequences comprising:
 providing a first nucleic acid sequence, and   applying a hybridization composition according to any of the  claims 1  to  26  to said first nucleic acid sequence for at least a time period sufficient to hybridize the first nucleic acid sequence and a second nucleic acid sequence.   
     
     
         31 . The method according to any one of the  claims 27  to  30 , wherein a sufficient amount of energy to hybridize the first and second nucleic acids is provided. 
     
     
         32 . The method according to  claim 31 , wherein the energy is provided by heating the hybridization composition and nucleic acid sequence. 
     
     
         33 . The method according to  claim 32 , wherein the heating step is performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating or heat lamps. 
     
     
         34 . The method according to any one of the  claims 27  to  33 , wherein the first nucleic acid sequence is double stranded and the second nucleic acid is single stranded. 
     
     
         35 . The method according to any one of the  claims 27  to  34 , wherein the denaturation and hybridization steps occur separately. 
     
     
         36 . The method according to any one of the  claims 27  to  35 , wherein the step of hybridization includes the steps of heating and cooling the hybridization composition and nucleic acid sequences. 
     
     
         37 . The method according to any one of the  claims 27  to  36 , wherein the step of hybridization takes less than 4 hours. 
     
     
         38 . The method according to  claim 37 , wherein the step of hybridization takes less than 2 hours. 
     
     
         39 . The method according to  claim 38 , wherein the step of hybridization takes less than 1 hour. 
     
     
         40 . The method according to  claim 39 , wherein the step of hybridization takes less than 30 minutes. 
     
     
         41 . The method according to  claim 40 , wherein the step of hybridization takes less than 5 minutes. 
     
     
         42 . The method according to any one of the  claims 27  to  41 , wherein the denaturation step is performed at 72 to 92° C. 
     
     
         43 . The method according to any one of the  claims 27  to  41 , wherein the denaturation step is performed at 75 to 95° C. 
     
     
         44 . The method according to any one of the  claims 27  to  43 , wherein the denaturation step is performed at 85° C. 
     
     
         45 . The method according to any one of the  claims 27  to  43 , wherein the denaturation step is performed at 82° C. 
     
     
         46 . The method according to any one of the  claims 27  to  45 , wherein the denaturation step takes less than 15 minutes. 
     
     
         47 . The method according to  claim 46 , wherein the denaturation step takes less than 5 minutes. 
     
     
         48 . The method according to any one of the  claims 27  to  47 , wherein the first nucleic acid sequence is in a biological sample. 
     
     
         49 . The method according to  claim 48 , wherein the biological sample is a cytology or histology sample. 
     
     
         50 . The method according to any one of the  claims 27  to  49 , further comprising a blocking step. 
     
     
         51 . Use of the hybridization composition according to any one of the  claims 1  to  26  for the molecular examination of DNA and RNA in the fields of cytology, histology, and molecular biology.