Home Applications Cloning & Synthetic Biology DNA End Modification Blunting

Blunting

Blunting is a process by which the single-stranded overhang created by a restriction digest is either "filled in", by the addition of nucleotides on the complementary strand using the overhang as a template for polymerization, or by "chewing back" the overhang, using an exonuclease activity. Vectors and inserts are often "blunted" to allow non-compatible ends to be joined. Sequence information is lost or distorted by doing this, and a detailed understanding of the modification should be sought before performing this procedure. Often, as long as the sequence being altered is not part of a translated region or a critical regulatory element, the consequence of creating blunt ends is negligible. Blunting a region of translated coding sequence, however, usually creates a shift in the reading frame. DNA polymerases, such as the Klenow fragment of DNA Polymerase I and T4 DNA Polymerase are often used to fill in (5´ → 3´) and chew back (3´ → 5´). Removal of a 5' overhang can be accomplished with a nuclease, such as Mung Bean Nuclease.

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Protocols for Blunting
Common Applications of Exonucleases and Endonucleases
NEB provides a list of common applications for our exonucleases and endonucleases.
Properties of Exonucleases and Endonucleases
NEB supplies many nucleases; several characteristics should be considered when choosing the one best suited to your particular research needs.
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Videos

  • DNABluntingVideo_thumb

    DNA Blunting Tutorial

    The first step in determining how your ends will be blunted is to determine if they are 5´ or 3´ overhangs. This tutorial will teach you how to identify what type of overhang you have, as well as which enzyme will blunt that end, and how.

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