Reducing Template Independent Primer Extension and Threshold Time for Loop Mediated Isothermal Amplification

Abstract

Compositions and methods are provided for loop mediated isothermal amplification in which single stranded binding proteins are shown to protect primers from non-specific extension and to stimulate the rate of threshold amplification.

What is claimed is: 
     
         1 . A preparation, comprising: a single stranded binding protein (SSB); a thermostable polymerase; at least four oligonucleotide primers, and a buffer. 
     
     
         2 . A preparation according to  claim 1 , wherein the buffer has a pH in the range of pH6-pH9, and optionally a stabilization agent selected from the group consisting of BSA, glycerol and detergent. 
     
     
         3 . A preparation according to  claim 1  wherein the buffer comprises a monovalent salt having a concentration in the range of 0-500 mM. 
     
     
         4 . A preparation according to  claim 1  wherein the buffer comprises a divalent metal cation having a concentration of 0.5 mM-10 mM. 
     
     
         5 . A preparation according to  claim 1 , wherein the buffer has a pH in the range of pH6-pH9, a monovalent salt having a concentration in the range of 0-500 mM, a divalent metal cation having a concentration of 0.5 mM-10 mM and optionally a stabilization agent selected from the group consisting of BSA, glycerol and detergent. 
     
     
         6 . A preparation according to  claim 1 , wherein the SSB is an extreme thermophile single strand binding protein (ET SSB). 
     
     
         7 . A preparation according to  claim 1 , wherein the thermostable polymerase has strand displacement activity and is active at temperatures of greater than 50° C.