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Abstract

The present disclosure relates, according to some embodiments, to immobilized enzyme compositions and methods for cleaving polynucleotide molecules including, for example, double-stranded DNA. Immobilized enzymes may comprise, for example, an enzyme (e.g., a type IIS restriction endonuclease, an RNAP, a capping enzyme), a support (e.g., a magnetic bead), and optionally, a linker disposed between the enzyme and the support. In some embodiments, methods may include contacting an immobilized enzyme with a polynucleotide substrate to form reaction products, separating the immobilized enzyme from the reaction products, and optionally reusing the immobilized enzymes in one or more subsequent reactions. preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase.

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First Claim