Thermostable blunt-end ligase and methods of use

Abstract

Fusion proteins having thermostable blunt-end ligase activity are provided. Blunt-end ligases are useful for DNA amplification, sequencing, production of recombinant DNA and recombinant fusion proteins, and other purposes. These thermostable blunt-end DNA ligases are useful in ligation schemes which include, e.g., an incubation at about 60-65° C. or higher, or as high as about 94° C., or at other temperatures. The ligases disclosed herein may enable high temperature blunt-end ligation without need for molecular crowding agents, and so may be useful for many nucleic acid ligation-amplification schemes, e.g., ones which operate at a uniform temperature (e.g., at about 60° C. or higher), including ones which require temperature cycling, e.g., from about 94° C. to about 60° C. (or higher) for one, two, three, or more cycles. The thermostable blunt-end DNA ligases disclosed herein enable higher specificity target amplification, for example, by permitting temperature denaturation of double-stranded DNA templates as well as specific primer binding.

1 - 7 . (canceled) 
     
     
         8 . A device for analyzing a sample containing DNA, comprising a thermostable blunt-end DNA ligase comprising a fusion protein comprising a DNA ligase and a DNA binding protein, wherein said thermostable blunt-end DNA ligase is suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher. 
     
     
         9 . The device of  claim 8 , wherein said thermostable blunt-end DNA ligase is selected from one of the following: a) the thermostable blunt-end DNA ligase of  claim 8  further comprising a T4 DNA ligase with an N-terminal p50 fusion and b) the thermostable blunt-end DNA ligase of  claim 8  comprising the amino acid sequence: 
       
         
           
                 
               
                   (SEQ ID NO: 8) 
                 
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