Method, kit and apparatus for the isolation of nucleic acids

Abstract

The invention concerns a process for preparing biological samples for the subsequent detection of an analyte. In particular, the invention relates to a process for the isolation of a nucleic acid in a sample using a suspension of magnetic glass particles. In addition, kits and apparatuses containing magnetic glass particles for sample preparation are provided.

1 . Process for the isolation of an analyte from a biological sample comprising the steps: 
 (a) lysing the sample in a reaction vessel,    (b) adding a solid adsorption matrix    (c) incubating under such conditions that the analyte binds to the adsorption matrix,    (d) removing non-bound sample components from the reaction vessel,    (e) incubating under such conditions that the analyte is eluted from the adsorption matrix and    (f) separating the eluate from the adsorption matrix.    
     
     
         2 . Process as claimed in  claim 1 ,  
       wherein 
 step (a) comprises adding a protease and a denaturing buffer.  
 
     
     
         3 . Process as claimed in  claim 2 ,  
       wherein 
 proteinase K is used as the protease.  
 
     
     
         4 . Process as claimed in  claim 2 ,  
       wherein 
 a denaturing buffer is used which contains a guanidinium salt, in particular guanidinium hydrochloride or/and guanidinium thiocyanate.  
 
     
     
         5 . Process as claimed in one of the previous claims,  
       wherein 
 magnetic glass particles are used as the solid adsorption matrix.  
 
     
     
         6 . Process as claimed in  claim 5 ,  
       wherein 
 the magnetic glass particles are added in the form of a suspension.  
 
     
     
         7 . Process as claimed in  claim 5  or  6 ,  
       wherein 
 glass particles are used whose glass phase contains SiO 2 , B 2 O 3  and Na 2 O or SiO 2 , B 2 O 3 , Al 2 O 3 , CaO and K 2 O.  
 
     
     
         8 . Process as claimed in one of the previous claims,  
       wherein 
 the amount of adsorption matrix added is at most 50% more than the amount that is required to quantitatively bind the analyte present in the sample.  
 
     
     
         9 . Process as claimed in one of the previous claims,  
       wherein 
 a continuous or intermittent mixing without adding external devices is carried out at least during steps (c), (d) or/and (e).  
 
     
     
         10 . Process as claimed in  claim 9 ,  
       wherein 
 the mixing is achieved by rotating the reaction vessel around its longitudinal axis.  
 
     
     
         11 . Process as claimed in claims  9  or  10 ,  
       wherein 
 the maximum period for carrying out steps (c) or/and (e) is 20 min in each case.  
 
     
     
         12 . Process as claimed in one of the previous claims,  
       wherein 
 step (d) comprises adding and aspirating a wash buffer which is optionally repeated several times.  
 
     
     
         13 . Process as claimed in  claim 12 ,  
       wherein 
 a wash buffer is used with a content of at least 50% (v/v) of an organic solvent that is miscible with water.  
 
     
     
         14 . Process as claimed in one of the previous claims,  
       wherein 
 additional reagents such as enzymes are added in step (e).  
 
     
     
         15 . Process as claimed in one of the previous claims,  
       wherein 
 a low salt buffer is used for elution in step (e).  
 
     
     
         16 . Process as claimed in one of the  claims 1  to  14 ,  
       wherein 
 a nucleic acid amplification master mix is added for elution in step (e).  
 
     
     
         17 . Process as claimed in one of the previous claims,  
       wherein 
 at least steps (c) and (d) are carried out at essentially the same temperature.  
 
     
     
         18 . Process as claimed in  claim 17 ,  
       wherein 
 step (a) or/and step (e) are also carried out at essentially the same temperature.  
 
     
     
         19 . Process as claimed in claims  17  or  18 ,  
       wherein 
 the temperature is in the range from room temperature to 40° C.  
 
     
     
         20 . Process as claimed in one of the  claims 17  to  19 ,  
       wherein 
 the temperature is in the range of 18° C. to 32° C.  
 
     
     
         21 . Process as claimed in one of the previous claims,  
       wherein 
 step (a) or/and step (e) are carried out at an elevated temperature.  
 
     
     
         22 . Process as claimed in one of the previous claims,  
       wherein 
 an aftertreatment step at an elevated temperature is carried out after step (f).  
 
     
     
         23 . Process as claimed in claims  21  or  22 ,  
       wherein 
 the elevated temperature is in the range from more than 40° C. to 95° C.  
 
     
     
         24 . Process for the isolation of an analyte from a biological sample comprising the steps: 
 (a) lysing the sample in a reaction vessel,    (b) adding a solid adsorption matrix    (c) incubating under such conditions that the analyte binds to the adsorption matrix,    (d) separating non-bound sample components from the adsorption matrix,    (e) incubating under such conditions that the analyte is eluted from the adsorption matrix and    (f) separating the eluate from the adsorption matrix    wherein at least steps (c) and (d) are carried out at essentially the same temperature.    
     
     
         25 . Process as claimed in one of the previous claims,  
       wherein 
 the analyte is a nucleic acid.  
 
     
     
         26 . Process as claimed in one of the previous claims,  
       wherein 
 the process is carried out in an automated device.  
 
     
     
         27 . Process as claimed in claims  1  or  24 ,  
       wherein 
 steps (a) to (e) are carried out in a single reaction vessel.  
 
     
     
         28 . Reagent kit in particular to carry out the process as claimed in one of the  claims 1  to  27 , comprising 
 (a) a protease,  
 (b) a sample lysing buffer,  
 (c) a wash buffer,  
 (d) an elution buffer and  
 (e) a suspension of magnetic glass particles.  
 
     
     
         29 . Reagent kit for the isolation of DNA comprising magnetic glass particles whose glass phase contains SiO 2 , B 2 O 3  and Na 2 O.  
     
     
         30 . Reagent kit for the isolation of RNA comprising magnetic glass particles whose glass phase contains SiO 2 , B 2 O 3 , Al 2 O 3 , CaO and K 2 O.  
     
     
         31 . Device for isolating an analyte from a biological sample comprising: 
 a sample preparation device ( 1 ),    a holding device for reagents ( 2 ),    a first holding device for reaction vessels for sample preparation ( 3 ) which is equipped for an operating temperature of ≦70° C., in particular ≦40° C.,    a second holding device for reaction vessels ( 4   a ,  4   b ,  4   c ), which optionally contains a cooling or/and heating means,    and a robotic tool device ( 5 ).    
     
     
         32 . Device as claimed in  claim 31 ,  
       wherein 
 a single reaction vessel is used to lyse a sample, to adsorb the analyte to a solid adsorption matrix, to wash the adsorption matrix and to elute the analyte from the adsorption matrix.  
 
     
     
         33 . Device as claimed in one of the claims  31  or  32 ,  
       wherein 
 the first holding device is used to hold the reaction vessels for the sample preparation and at least for the adsorption of the analyte to a solid adsorption matrix and for washing the adsorption matrix.  
 
     
     
         34 . Device as claimed in  claim 33 ,  
       wherein 
 the first holding device is addition ally used to hold the reaction vessels for sample preparation, for lysing the sample or/and for eluting the analyte from the adsorption matrix.  
 
     
     
         35 . Device as claimed in one of the  claims 31  to  34 ,  
       wherein 
 the second holding device for holding reaction vessels is used to store or/and further process the analyte.  
 
     
     
         36 . Device as claimed in one of the  claims 1  to  35 ,  
       wherein 
 the second holding device for holding vessels for reagents is used to process the sample further.  
 
     
     
         37 . Device as claimed in one of the  claims 31  to  36 ,  
       wherein 
 the second holding device for holding reaction vessels is used for at least one treatment step at an elevated temperature which is selected from lysing the sample, eluting the sample from the adsorption matrix and an aftertreatment step after elution.  
 
     
     
         38 . Device as claimed in one of the  claims 31  to  37 ,  
       wherein 
 the first holding device contains means for magnetic separation.  
 
     
     
         39 . Device as claimed in one of the  claims 31  to  38 ,  
       wherein 
 the first holding device contains means for mixing the reaction vessels by rotation around their longitudinal axis.  
 
     
     
         40 . Device as claimed in one of the  claims 31  to  39 ,  
       wherein 
 the robotic tool comprises automatic pipetting devices and optionally means for opening and closing reaction vessels.  
 
     
     
         41 . Device as claimed in one of the  claims 31  to  40 ,  
       wherein 
 the robotic tool comprises means for transporting reaction vessels between the first and second holding device.  
 
     
     
         42 . Magnetic glass particles comprising a magnetic core and a glass coat which contains SiO 2 , B 2 O 3 , an alkali metal oxide and optionally Al 2 O 3  and an alkaline earth metal oxide.  
     
     
         43 . Glass particles as claimed in  claim 41 ,  
       wherein 
 the glass coat contains SiO 2 , B 2 O 3  and Na 2 O or SiO 2 , B 2 O 3 , Al 2 O 3 , K 2 O and CaO.