{"id":8731,"date":"2015-06-17T20:31:16","date_gmt":"2015-06-17T20:31:16","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/behindthebench\/?p=8731"},"modified":"2020-09-26T00:12:07","modified_gmt":"2020-09-26T00:12:07","slug":"how-does-sanger-sequencing-work","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/how-does-sanger-sequencing-work\/","title":{"rendered":"How Does Sanger Sequencing Work?"},"content":{"rendered":"<p>Let\u2019s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it \u2013 we\u2019re talking about\u00a0<a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/sanger_sequencing_method.html?icid=lsg_gsd_CP1236_PJT8083_blog_seqitout-howsangerworks\" target=\"_blank\" rel=\"noopener noreferrer\">Sanger Sequencing<\/a>\u00a0by capillary electrophoresis. Many might ask, \u201cwhy is it called Sanger Sequencing?\u201d\u00a0Sanger Sequencing is named after the inventor of this ground breaking technology, Dr.\u00a0<a href=\"https:\/\/en.wikipedia.org\/?title=Frederick_Sanger\" target=\"_blank\" rel=\"nofollow noopener noreferrer\">Frederick Sanger<\/a>, who developed this method over 40 years ago in the mid-70s.\u00a0So, what are the basics of Sanger Sequencing?<\/p>\n<section class=\"jive-content-body\">\n<div class=\"jive-blog-post-message\">\n<div class=\"jive-rendered-content\">\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/e2G5zx-OJIw?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p>It all starts by having a short primer binding next to the region of interest. In the presence of the 4 nucleotides, the polymerase will extend the primer by adding on the complementary nucleotide from the template DNA strand. To find the exact composition of the DNA sequence, we need to bring this reaction to a defined stop that allows us to identify the base of the very end of this particular DNA fragment. Sanger did this by removing an oxygen atom from the ribonucleotide. Such a nucleotide is called a dideoxynucleotide. This is analogous to throwing a wrench into a gear. The polymerase enzyme can no longer add normal nucleotides onto this DNA chain. The extension has stopped and we now need to identify what it is. We identify the chain terminating nucleotide by a specific fluorescent dye, 4 specific colors to be exact. Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3\u2032 end.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-8733 aligncenter\" src=\"http:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM.png\" alt=\"How does Sanger sequencing work?\" width=\"828\" height=\"464\" srcset=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM.png 1277w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM-300x168.png 300w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM-768x430.png 768w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM-1024x573.png 1024w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM-1250x700.png 1250w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM-400x224.png 400w\" sizes=\"auto, (max-width: 828px) 100vw, 828px\" \/><\/p>\n<p>The extension products are then separated by\u00a0<a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing.html?icid=lsg_gsd_CP1236_PJT8083_blog_seqitout-howsangerworks\" target=\"_blank\" rel=\"noopener noreferrer\">Capillary Electrophoresis<\/a>\u00a0or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer. During CE, an electrical field is applied so that the negatively charged DNA fragments move toward the positive electrode. The speed at which a DNA fragment migrates through the medium is inversely proportional to its molecular weight. This process can separate the extension products by size at a resolution of one base. A laser excites the dye labeled DNA fragments as they pass through a tiny window at the end of the capillary. The excited dye emits a light at a characteristic wavelength that is detected by a light sensor. Software can then interpret the detected signal and translate it into a base call. When the sequencing reaction is performed in the presence of all four terminated nucleotides, you eventually get a pool of DNA fragments that are measured and separated base by base. What you will get in the end is a data file showing the sequence of the DNA in a colorful electropherogram and a text file which you can use to answer the questions you may be asking.<\/p>\n<p>And that, in a nutshell is Sanger Sequencing.<\/p>\n<p>If you want to learn more, just download our free\u00a0<a href=\"https:\/\/tools.thermofisher.com\/content\/sfs\/manuals\/cms_041003.pdf?icid=lsg_gsd_CP1236_PJT8083_blog_seqitout-howsangerworks\" target=\"_blank\" rel=\"noopener noreferrer\">Sanger Sequencing Handbook<\/a>.<\/p>\n<p>We\u2019re making new Seq It Out videos all the time, so if you have an idea the would make a great topic,\u00a0<a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/products-and-services\/promotions\/ask.html?icid=lsg_gsd_CP1236_PJT8083_blog_seqitout-howsangerworks\" target=\"_blank\" rel=\"noopener noreferrer\">just drop us a line<\/a>.<\/p>\n<\/div>\n<\/div>\n<\/section>\n","protected":false},"excerpt":{"rendered":"<p>Let\u2019s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it \u2013 we\u2019re talking about\u00a0Sanger Sequencing\u00a0by capillary electrophoresis. Many might ask, \u201cwhy is it called Sanger Sequencing?\u201d\u00a0Sanger Sequencing is named after the inventor of this ground breaking technology, Dr.\u00a0Frederick Sanger, who<\/p>\n","protected":false},"author":111,"featured_media":8733,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[108],"tags":[17,36,99,25,176,74],"division":[],"class_list":{"0":"post-8731","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"tag-applied-biosystems","9":"tag-capillary-electrophoresis","10":"tag-fragment-analysis","11":"tag-sanger-sequencing","12":"tag-seqitout","13":"tag-sequencing","14":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>How Does Sanger Sequencing Work? - Behind the Bench<\/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.thermofisher.com\/blog\/behindthebench\/how-does-sanger-sequencing-work\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"How Does Sanger Sequencing Work?\" \/>\n<meta property=\"og:description\" content=\"Let\u2019s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it \u2013 we\u2019re talking about\u00a0Sanger Sequencing\u00a0by capillary electrophoresis. Many might ask, \u201cwhy is it called Sanger Sequencing?\u201d\u00a0Sanger Sequencing is named after the inventor of this ground breaking technology, Dr.\u00a0Frederick Sanger, who\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/how-does-sanger-sequencing-work\/\" \/>\n<meta property=\"og:site_name\" content=\"Behind the Bench\" \/>\n<meta property=\"article:publisher\" content=\"https:\/\/www.facebook.com\/thermofisher\" \/>\n<meta property=\"article:published_time\" content=\"2015-06-17T20:31:16+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2020-09-26T00:12:07+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/06\/Screen-Shot-2016-06-03-at-1.35.20-PM.png\" \/>\n\t<meta property=\"og:image:width\" content=\"1277\" \/>\n\t<meta property=\"og:image:height\" content=\"715\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/png\" \/>\n<meta name=\"author\" content=\"Jeremy Schoales\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:creator\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:site\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"Jeremy Schoales\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/#article\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/\"},\"author\":{\"name\":\"Jeremy Schoales\",\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/behindthebench\\\/#\\\/schema\\\/person\\\/01e15b61daaa9db3ec4d5fcb2e9367ee\"},\"headline\":\"How Does Sanger Sequencing Work?\",\"datePublished\":\"2015-06-17T20:31:16+00:00\",\"dateModified\":\"2020-09-26T00:12:07+00:00\",\"mainEntityOfPage\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/\"},\"wordCount\":506,\"commentCount\":5,\"image\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/#primaryimage\"},\"thumbnailUrl\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/behindthebench\\\/wp-content\\\/uploads\\\/sites\\\/9\\\/2016\\\/06\\\/Screen-Shot-2016-06-03-at-1.35.20-PM.png\",\"keywords\":[\"Applied Biosystems\",\"Capillary Electrophoresis\",\"Fragment Analysis\",\"Sanger Sequencing\",\"SeqitOut\",\"sequencing\"],\"articleSection\":[\"Lab Tips and Tricks\"],\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"CommentAction\",\"name\":\"Comment\",\"target\":[\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/#respond\"]}]},{\"@type\":\"WebPage\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/\",\"url\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/how-does-sanger-sequencing-work\\\/\",\"name\":\"How Does Sanger Sequencing Work? 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