Integrated sample preparation systems and stabilized enzyme mixtures

Abstract

The present invention provides integrated sample preparation systems and stabilized enzyme mixtures. In particular, the present invention provides microfluidic cards configured for processing a sample and generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other suitable nucleic acid analysis methods. The present invention also provides stabilized enzyme mixtures containing an enzyme (e.g., an enzyme used in whole genome amplification), BSA, and a sugar. Such enzyme mixtures may be lyophilized and stored at room temperature without significant loss of enzyme activity for months.

We claim: 
     
         1 . A stabilized enzyme mixture comprising:
 a) at least one type of enzyme;   b) bovine serum albumin (BSA);   c) a sugar;   d) an inorganic salt;   e) a divalent metal cation;   f) a buffering agent;   g) an emulsifier; and   h) a reducing agent.   
     
     
         2 . The stabilized enzyme mixture of  claim 1 , further comprising polyethylene glycol (PEG). 
     
     
         3 . The stabilized enzyme mixture of  claim 1 , wherein said stabilize enzyme mixture is in a lyophilized form. 
     
     
         4 . The stabilized enzyme mixture of  claim 1 , wherein said BSA is present at a concentration of 0.05%-3.0% in said stabilized enzyme mixture. 
     
     
         5 . The stabilized enzyme mixture of  claim 1 , wherein said sugar is present at a concentration of 5-35% of said stabilized enzyme mixture. 
     
     
         6 . The stabilized enzyme mixture of  claim 1 , wherein said sugar is a non-reducing sugar. 
     
     
         7 . The stabilized enzyme mixture of  claim 1 , wherein said at least one type of enzyme comprises a polymerase. 
     
     
         8 . The stabilized enzyme mixture of  claim 1 , wherein said at least one type of enzyme comprises an inorganic pyrophosphatase. 
     
     
         9 . The stabilized enzyme mixture of  claim 1 , wherein said inorganic salt is present at a concentration of 1 mM-25 mM in said stabilized enzyme mixture. 
     
     
         10 . The stabilized enzyme mixture of  claim 1 , wherein divalent metal cation is present at a concentration of 1 mM-30 mM in said stabilized enzyme mixture. 
     
     
         11 . The stabilized enzyme mixture of  claim 1 , wherein said buffering agent is present at a concentration of 10 mM-100 mM. 
     
     
         12 . The stabilized enzyme mixture of  claim 1 , wherein said emulsifier is present at a concentration of 0.01%-0.15% in said stabilized enzyme mixture. 
     
     
         13 . The stabilized enzyme mixture of  claim 1 , wherein said reducing agent is present at a concentration of 1 mM-10 mM.