{"id":73,"date":"2023-04-13T17:55:56","date_gmt":"2023-04-13T17:55:56","guid":{"rendered":"https:\/\/test-hcc-press-wp-multisite.pantheonsite.io\/bsc2010l\/?post_type=chapter&p=73"},"modified":"2025-10-29T16:46:59","modified_gmt":"2025-10-29T16:46:59","slug":"chapter-11-dna-gel-electrophoresis","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hccfl.edu\/Bio1LabManual\/chapter\/chapter-11-dna-gel-electrophoresis\/","title":{"raw":"Chapter 10 - DNA Gel Electrophoresis","rendered":"Chapter 10 – DNA Gel Electrophoresis"},"content":{"raw":"

DNA Gel Electrophoresis<\/h2>\r\n

BACKGROUND<\/h3>\r\nWhen first introduced, DNA fingerprinting (DNA profile analysis or DNA typing) involved the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes. In contrast to more conventional methodologies, such as blood typing, which excludes suspects, traditional DNA fingerprinting provides accurate, unambiguous identification of the source of DNA samples. Variations in DNA sequences between individuals, as determined by differences in restriction enzyme cleavage patterns, are known as <\/span>[pb_glossary id=\"645\"]restriction fragment length polymorphisms[\/pb_glossary]. RFLPs are a manifestation of the unique molecular genetic profile, or \"fingerprint,\" of an <\/span>individual's DNA.<\/span>\r\n\r\n[pb_glossary id=\"624\"]Restriction enzymes[\/pb_glossary]\u00a0<\/span><\/strong>are endonucleases that catalyze the cleavage of phosphate bonds. They require Mg2+ for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the cleavage point. The distinguishing feature of restriction enzymes compared to other endonucleases is that they only cut at specific sequences of bases.<\/span>\r\n\r\n[caption id=\"attachment_634\" align=\"aligncenter\" width=\"238\"]\"Examples Examples of Restriction Enzymes.[\/caption]\r\n\r\nMany different species of bacteria produce restriction enzymes, including cyanobacteria (blue-green algae). Over 3,000 restriction enzymes have been discovered and cataloged.\u00a0<\/span>\r\n\r\nA [pb_glossary id=\"624\"]restriction enzyme[\/pb_glossary] requires a specific double-stranded recognition sequence of nucleotide bases to cut DNA. Recognition sites are generally 4 to 8 base pairs in length, and cleavage occurs within or near that site.<\/span>\r\n\r\nRecognition sites are frequently symmetrical, i.e., both DNA strands in the site have the same base sequence when read 5' to 3'. Such sequences are called\u00a0<\/span>[pb_glossary id=\"642\"]palindromes[\/pb_glossary].\u00a0<\/span><\/strong>\r\n\r\n[pb_glossary id=\"649\"]Agarose gels[\/pb_glossary] are easily cast and handled compared to other matrices because the gel setting is a physical rather than chemical change. Samples can also be recovered (removed) from the gel and used in downstream applications.<\/span>\r\n\r\nThe technique separates DNA fragments ranging from 50 base pair to several megabases (millions of bases). The distance between DNA bands of a given length is determined by the percent agarose in the gel.<\/span>\r\n\r\nMost agarose gels are made with between 0.7% (good separation or resolution of large 5\u201310kb DNA fragments) and 2% agarose (good resolution for small 0.2\u20131kb fragments) dissolved in electrophoresis buffer. For most applications or analyses of unknown DNA sizes, a 1% gel is effective.<\/span>\r\n\r\nThe DNA is always added to the electrically Negative (black) side of the electric current. DNA's natural negative charge allows it to be pulled through the gel toward the positive (red) end. Remember, DNA \"RUNS TO RED.\"\r\n\r\nThe agarose gel acts as a molecular sieve in that larger pieces of DNA migrate more slowly than smaller pieces. Thus, after the technique, larger fragments of DNA are closer to the wells from which they originated, and smaller fragments travel further into the gel.<\/span>\r\n\r\nA\u00a0<\/span>[pb_glossary id=\"647\"]size marker[\/pb_glossary](a DNA ladder or Molecular Weight Marker) is commonly run in a well adjacent to the analyzed samples to make the migration distances more informative. Size markers contain DNA fragments with known <\/span>DNA sizes. So if your band migrates the same distance as one of the bands from the size marker, it's safe to say it is approximately the same size.<\/span>\r\n\r\nHow the DNA migrates in the gel depends on the DNA fragment's size and the DNA strand's orientation. Double-stranded DNA fragments behave like long rods, so their migration through the gel is relative to their size. Circular DNA, such as plasmids, may show multiple bands or even a smear because the migration speed may depend on whether it is relaxed or\u00a0<\/span>supercoiled<\/span><\/strong>. Supercoiling is a molecular conformation in which circular DNA is wrapped and tightly coiled around itself, thus taking up less space than a relaxed piece of DNA.<\/span>\r\n\r\nAdditional Resources for Gel Electroresis\r\n