{"id":1878,"date":"2015-04-24T10:38:02","date_gmt":"2015-04-24T10:38:02","guid":{"rendered":"http:\/\/www.neb-online.de\/?page_id=1878"},"modified":"2020-07-31T07:23:25","modified_gmt":"2020-07-31T05:23:25","slug":"dna-modifying-enzymes","status":"publish","type":"page","link":"https:\/\/www.neb-online.de\/en\/cloning-synthetic-biology\/dna-modifying-enzymes\/","title":{"rendered":"DNA Modifying Enzymes"},"content":{"rendered":"

[vc_row][vc_column][vc_basic_grid post_type=”post” max_items=”10″ element_width=”12″ gap=”15″ item=”4123″ taxonomies=”72″ grid_id=”vc_gid:1596172909916-fa0b5fa7-7939-8″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

DNA\u00a0Modification<\/h2>\n

Producing DNA samples by shearing, nebulization, restriction enzyme digestion<\/a> or PCR amplification<\/a> frequently leaves DNA molecules with ends incompatible for downstream experiments. Selective enzymatic treatment is used to prepare DNA for ligation.<\/p>\n

Ligation<\/a>, the subsequent step to DNA end modification in the cloning<\/a> process, is the formation of covalent\u00a0phosphodiester bonds\u00a0between\u00a0the 3′ hydroxyl and 5\u2019 phosphate ends\u00a0of DNA and the vector. Preparation of DNA for vector ligation can include different types of end treatments, such as end-blunting, phosphorylation and dephosphorylation. Some enzymes couple the removal of the 3\u2019 phosphate to expose hydroxyl groups, and the addition of a phosphate group to the 5\u2019 end.\u00a0Ligation\u00a0of DNA with other molecules can either be through complementary sequence base pairing or blunt-ended ligation. The coupled action of enzymes can be used to remove any terminal single-stranded overhangs that are left after DNA digestion by restriction endonuclease or added to DNA during PCR amplification.<\/p>\n

These end treatments can also be used to alter the phosphorylation state of the 5\u2019 end of DNA for detection, isolation and sequencing applications. Many investigations use the addition of a phosphate radioisotope for labeling applications.[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”custom” accent_color=”#eb7e2d”][\/vc_column][\/vc_row][vc_row][vc_column width=”1\/2″][vc_column_text]<\/p>\n

Dephosphorylation<\/h2>\n

Digested DNA typically possesses a 5\u2019 phosphate group that is required for ligation. In order to prevent self-ligation, the 5′ phosphate can be removed prior to ligation. Dephosphorylation of the 5\u2019 end prohibits self-ligation, enabling the researcher to manipulate the DNA as desired before re-ligating. Generally, it is a good idea to dephosphorylate the linearized vector to facilitate ligation of recombinant DNA molecules.\u00a0 In fact, vector self-ligation increases the background activity of the cloning process.[\/vc_column_text][\/vc_column][vc_column width=”1\/2″][vc_text_separator title=”Video: DNA Dephosphorylation” color=”custom” accent_color=”#eb7e2d”][vc_video link=”https:\/\/youtu.be\/L0DVW3O–kM”][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Our recommended phosphatase for DNA dephosphorylation is the Quick CIP #M0525<\/a>.
\nQuick CIP is a heat-labile version of calf intestinal alkaline phosphatase (CIP) purified from a recombinant source.<\/p>\n