Creation of a type IIS restriction endonuclease with a long recognition sequence |
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| Authors: | Shaun M Lippow Patti M Aha Matthew H Parker William J Blake Brian M Baynes and Da&#x;a Lipov&#x;ek |
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| Institution: | Shaun M. Lippow, Patti M. Aha, Matthew H. Parker, William J. Blake, Brian M. Baynes, and Daša Lipovšek |
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| Abstract: | Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 | |
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