Nucleic acid isolation

Abstract

The present invention relates to a method of isolating nucleic acid from a blood sample, said method comprising: (a) selectively isolating leucocytes from said sample by binding said leucocytes to a solid support by means of a binding partner specific for leucocytes; (b) lysing said isolated leucocytes; and (c) binding nucleic acid released from said lysed cells to said solid support. Kits for isolating nucleic acid from samples form further embodiments of the invention.

1 . A method of isolating nucleic acid from a blood sample, said method comprising: 
 (a) selectively isolating leucocytes from said sample by binding said leucocytes to a solid support by means of a binding partner specific for leucocytes;    (b) lysing said isolated leucocytes; and    (c) binding nucleic acid released from said lysed cells to said solid support.    
     
     
         2 . The method of  claim 1  wherein in step (a) leucocytes in said sample are bound to a leucocyte-specific binding partner, said binding partner being attached to a solid support before or after binding to said leucocytes, thereby to bind said support to said leucocytes.  
     
     
         3 . The method of  claim 1  or  claim 2  wherein the nucleic acid is DNA, RNA or any naturally occurring modification thereof, or combinations thereof.  
     
     
         4 . The method as claimed in any one of  claims 1  to  3  wherein the binding partner in step (a) binds specifically to leucocytes present in the sample but not to other cells or components of the sample.  
     
     
         5 . The method of  claim 4  wherein said binding partner is an antibody or a fragment or derivative of an antibody.  
     
     
         6 . The method of any one of  claims 1  to  5  wherein one or more different binding partners are used to isolate the leucocytes.  
     
     
         7 . The method of any one of  claims 1  to  6  wherein all or substantially all leucocytes present in the sample are separated.  
     
     
         8 . The method of any one of  claims 1  to  7  wherein the binding partner recognises or is capable of binding specifically to one or more of the molecules selected from the group comprising HLA-I, CD11a, CD18, CD45, CD46, CD50, CD82, CD100, CD162, CD5 and CD 15.  
     
     
         9 . The method of  claim 8  wherein one or more of said binding partners are used.  
     
     
         10 . The method of claims  8  or  claim 9  wherein a combination of-binding partners specific for CD45 and CD15 are used.  
     
     
         11 . The method of any one of  claims 1  to  10  wherein the solid support of step (a) comprises particles, preferably polymeric particles.  
     
     
         12 . The method of any one of  claims 1  to  11  wherein said solid support is magnetic, preferably superparamagnetic.  
     
     
         13 . The method of any one of the preceding claims wherein the binding partner is attached directly or indirectly to the solid support of step (a).  
     
     
         14 . The method of any one of  claims 9  to  13  wherein more than one different type of binding partner is used and said binding partners are attached to the same or different solid support.  
     
     
         15 . The method of any one of  claims 1  to  14  wherein the nucleic acid binding in step (c) is carried out using a detergent based system.  
     
     
         16 . The method of any one of  claims 1  to  15  wherein said isolated cells of step (b) or the nucleic acid of step (c) are additionally contacted with an additional amount of a second solid support, preferably binding said nucleic acid to said second solid support which is capable of binding nucleic acid.  
     
     
         17 . A method of isolating nucleic acid from a cell sample, said method comprising; 
 (a) selectively isolating cells from said sample by binding said cells to a solid support by means of one or more appropriate binding partners specific for said cells;    (b) lysing said isolated cells;    (c) binding nucleic acid released from said lysed cells to said first solid support; and    (d) contacting said isolated cells of step (b) or the nucleic acid of step (c) with an additional amount of a second solid support, preferably binding said nucleic acid to said second solid support which is capable of binding nucleic acid.    
     
     
         18 . The method of  claim 17  wherein the cells are leucocytes.  
     
     
         19 . The method of  claim 17  or  18  wherein the cell sample is a blood sample.  
     
     
         20 . The method of any one of  claims 17  to  19  wherein 30 the components used in, or features present in steps (a), (b) and (c) are as defined in any one of the preceding claims.  
     
     
         21 . The method of any one of  claims 17  to  20  wherein the isolated cells are lysed in the presence of said second solid support.  
     
     
         22 . The method of any one of  claims 18  to  21  wherein the first solid support is as defined in any one of  claims 8  to  10  and the second solid support which is different to said first solid support is negatively charged.  
     
     
         23 . The method of any one of  claims 1  to  22  wherein the isolated nucleic acid is DNA and the method comprises a further step to isolate RNA from the sample.  
     
     
         24 . A kit for isolating nucleic acid from a sample comprising: 
 (a) a solid support;    (b) means for binding leucocytes to said solid support;    (c) means for lysing said cells; and    (d) means for binding nucleic acid released from said lysed cells to said same solid support.    
     
     
         25 . The kit of  claim 24  further comprising one or more of: 
 (e) a second solid support;  
 (f) a proteinase; and  
 (g) means for detecting nucleic acid.  
 
     
     
         26 . A kit for isolating nucleic acid from a cell sample comprising: 
 (a) a solid support;    (b) means for binding the particular cells from which it is desired to isolate nucleic acid to said solid support;    (c) means for lysing said cells;    (d) means for binding nucleic acid released from said lysed cells to said same solid support; and    (e) a second solid support.    
     
     
         27 . The method of any one of  claims 1  to  23  wherein said method is performed using an automated system for handling of the solid support during cell lysis, nucleic acid binding and optionally washing steps.  
     
     
         28 . The method of  claim 27  wherein said automated system additionally handles the support during: the cell isolation stage.  
     
     
         29 . The method of  claim 27  or  claim 28  wherein the Bead Retriever™ apparatus is used.