{"id":1878,"date":"2015-04-24T10:38:02","date_gmt":"2015-04-24T10:38:02","guid":{"rendered":"http:\/\/www.neb-online.fr\/?page_id=1878"},"modified":"2020-04-10T15:00:49","modified_gmt":"2020-04-10T13:00:49","slug":"dna-modifying-enzymes","status":"publish","type":"page","link":"https:\/\/www.neb-online.fr\/en\/cloning-synthetic-biology\/dna-modifying-enzymes\/","title":{"rendered":"DNA Modifying Enzymes"},"content":{"rendered":"<div class=\"wpb-content-wrapper\"><p>[vc_row][vc_column][vc_column_text]<\/p>\n<h2>DNA\u00a0Modification<\/h2>\n<p>Producing DNA samples by shearing, nebulization, <a title=\"Restriction Enzymes\" href=\"http:\/\/www.neb-online.fr\/en\/neb-encloning-synthetic-biology\/restriktionsenzyme\/\">restriction enzyme digestion<\/a> or <a title=\"PCR and DNA Amplification\" href=\"http:\/\/www.neb-online.fr\/en\/neb-enpcr-and-dna-amplification\/\">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<p><a title=\"DNA Ligases\" href=\"http:\/\/www.neb-online.fr\/en\/neb-encloning-synthetic-biology\/dna-ligases\/\">Ligation<\/a>, the subsequent step to DNA end modification in the <a title=\"Cloning \/ Synthetic Biology\" href=\"http:\/\/www.neb-online.fr\/en\/neb-encloning-synthetic-biology\/\">cloning<\/a> process, is the formation of covalent\u00a0phosphodiester bonds\u00a0between\u00a0the 3&#8242; 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<p>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=&#8221;black&#8221;][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n<h2>Dephosphorylation<\/h2>\n<p>Digested DNA typically possesses a 5\u2019 phosphate group that is required for ligation. In order to prevent self-ligation, the 5&#8242; 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. Dephosphorylation can be accomplished using any of a number of phosphatases, including Shrimp Alkaline Phosphatase (rSAP) (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0371@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0371<\/a>),\u00a0Quick CIP Phosphatase (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0525@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0525<\/a>) and Antarctic Phosphatase (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0289@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0289<\/a>).[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=&#8221;black&#8221;][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n<h2>Blunting<\/h2>\n<p>Blunting is the elimination of incompatible 3\u2019 or 5\u2019 overhangs for the promotion of blunt-end ligation.\u00a0 Several approaches may be used for DNA end blunting. Terminal unpaired nucleotides may be removed from DNA ends by using an enzyme with exonuclease activity, which hydrolyzes a terminal phosphodiester bond, thereby removing the overhang one base at a time. DNA fragments with 5\u2019 overhangs may be blunted by filling in a recessed 3\u2019 terminus with DNA polymerase in the presence of dNTPs. End removal or fill-in can be accomplished using a number of enzymes, including DNA Polymerase I Large (Klenow) Fragment (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0210@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0210<\/a>), T4 DNA Polymerase (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0203@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0203<\/a>) or Mung Bean Nuclease (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0250@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0250<\/a>). Once blunted, DNA is universally compatible with other blunt-ended fragments and vectors.[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=&#8221;black&#8221;][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n<h2>Phosphorylation<\/h2>\n<p>Single- or double-stranded DNA with a 5&#8242;-hydroxyl terminus has to be phosphorylated prior to ligation as 5\u2019 phosphate groups are required for ligation. Phosporylation of DNA ends with phosphate radioisotopes can also be used to label DNA in preparation of DNA for subsequent detection, isolation and sequencing applications. A number of polynucleotide kinases, including T4 PNK (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0201@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0201<\/a>) and T4 PNK (3&#8242; phosphatase minus) (<a href=\"https:\/\/shop.neb-online.fr\/en\/art\/M0236@\" target=\"_blank\" rel=\"noopener noreferrer\">NEB #M0236<\/a>), can be used to transfer the \u03b3-phosphate of ATP to a 5\u2019 terminus of DNA.[\/vc_column_text][\/vc_column][\/vc_row]<\/p>\n<\/div>","protected":false},"excerpt":{"rendered":"<p>[vc_row][vc_column][vc_column_text] DNA\u00a0Modification Producing DNA samples by shearing, nebulization, restriction enzyme digestion or PCR amplification frequently leaves DNA molecules with ends incompatible for downstream experiments. Selective enzymatic treatment is used to prepare DNA for ligation. Ligation, the subsequent step to DNA end modification in the cloning process, is the formation of covalent\u00a0phosphodiester bonds\u00a0between\u00a0the 3&#8242; hydroxyl and&#8230;  <a class=\"excerpt-read-more\" href=\"https:\/\/www.neb-online.fr\/en\/cloning-synthetic-biology\/dna-modifying-enzymes\/\" title=\"Read DNA Modifying Enzymes\">Read more &raquo;<\/a><\/p>\n","protected":false},"author":15,"featured_media":1849,"parent":1824,"menu_order":0,"comment_status":"open","ping_status":"open","template":"page-sidebar-cloning-synthetic-biology.php","meta":{"footnotes":""},"class_list":["post-1878","page","type-page","status-publish","has-post-thumbnail","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.0 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>DNA Modifying Enzymes - New England Biolabs France<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.neb-online.fr\/en\/cloning-synthetic-biology\/dna-modifying-enzymes\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"DNA Modifying Enzymes - New England Biolabs France\" \/>\n<meta property=\"og:description\" content=\"[vc_row][vc_column][vc_column_text] DNA\u00a0Modification Producing DNA samples by shearing, nebulization, restriction enzyme digestion or PCR amplification frequently leaves DNA molecules with ends incompatible for downstream experiments. Selective enzymatic treatment is used to prepare DNA for ligation. Ligation, the subsequent step to DNA end modification in the cloning process, is the formation of covalent\u00a0phosphodiester bonds\u00a0between\u00a0the 3&#8242; hydroxyl and... 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Selective enzymatic treatment is used to prepare DNA for ligation. Ligation, the subsequent step to DNA end modification in the cloning process, is the formation of covalent\u00a0phosphodiester bonds\u00a0between\u00a0the 3&#8242; hydroxyl and... 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