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Sanger DNA Sequencing, From Then to Now.
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Sanger DNA Sequencing, From Then to Now.

Sanger Sequencing: Understanding the Method

Introduction to Sanger Sequencing

  • In 1977, Frederick Sanger introduced a method for DNA sequencing using chain-terminating inhibitors known as ddNTPs.
  • DNA consists of four nucleotides (dNTPs), which are deoxyribonucleoside triphosphates, essential for copying and growing DNA strands.

Structure of dNTPs and ddNTPs

  • A dNTP comprises one deoxyribose sugar, a base (A, T, C, or G), and three phosphate groups; while ddNTP has two fewer oxygens than ribose.
  • The role of DNA polymerase is to add complementary bases to the growing strand by catalyzing reactions that release phosphate groups.

Mechanism of Sanger Sequencing

  • The process involves understanding the 5' and 3' ends of nucleotides; DNA synthesis extends from the 3' end.
  • Original Sanger sequencing was manual and utilized radioactive dyes; it required specific components like primers, DNA polymerase, dNTPs, and ddNTPs.

Steps in Original Sanger Sequencing

  • The procedure starts with heating the mixture to separate DNA strands before cooling to allow primer binding.
  • Each tube contains a different ddNTP at lower concentrations leading to random termination at various lengths during extension.

Visualization and Base Calling

  • Fragments are separated on a polyacrylamide gel where size differentiation allows visualization after drying onto paper support.
  • "Base calling" reads sequences from shortest fragments first; this labor-intensive method took days for limited samples.

Advancements in Sequencing Technology

Transition to Automated Systems

  • Applied Biosystems launched the AB370A in 1987, marking a shift towards automation in sequencing with fluorescent dyes replacing radioactive ones.

Enhanced Efficiency with New Instruments

  • The AB370A could run multiple samples simultaneously with automated data processing through laser detection of fragments.

Implications for Human Genome Project

  • By 1990, only <2% of the human genome had been sequenced; advancements suggested full genome mapping was feasible due to improved technology.

Role of PCR in Sequencing

Sanger Sequencing: Innovations and Comparisons

Linear PCR and Cycle Sequencing

  • Primers are incorporated into DNA fragments, enhancing the fluorescent signal. However, only forward strands are produced, leading to a linear increase in fragment numbers with each cycle, termed linear PCR.
  • The advancement of capillary electrophoresis allows DNA to run through gel in fine tubes under electric current, detected by lasers. This method improves efficiency as heat escapes easily.

Capillary Electrophoresis Development

  • Beckman Coulter introduced the first commercial capillary electrophoresis instrument in 1989, which led to the creation of the ABI PRISM 310 Sanger sequencing system by Applied Biosystems in 1995.
  • The ABI PRISM 310 significantly reduced sample run time from 14 hours to under three hours while increasing sequencing length capability up to 600 base pairs.

Automation and Efficiency Improvements

  • Electrokinetic injection allows low volumes of DNA samples to be used; an electrical current pulls DNA into the capillary for separation based on size.
  • The introduction of BigDyeTerminators in 1997 allowed all sequencing reactions to occur in one tube rather than requiring four separate reactions with fluorescent primers.

Human Genome Project and Celera Genomics

  • In response to slow progress (only 6% sequenced by 1998), Applied Biosystems launched the ABI PRISM 3700 with 96 capillaries, partnering with TIGR led by Craig Venter to form Celera.
  • Celera aimed for rapid human genome sequencing for profit, causing controversy among scientists due to plans for gene patenting amidst competition with public efforts.

Impact on Genome Sequencing

  • Each run of the ABI PRISM 3700 could process up to 1,536 samples daily at a cost-effective rate while generating significant sequence data quickly.
  • Despite newer technologies like Next Generation Sequencing (NGS), Sanger remains the gold standard due to its high accuracy (99.9%) compared to NGS's variable accuracy depending on depth.

Cost-effectiveness and Sensitivity Comparison

  • Sanger is more cost-effective for fewer samples (<20), while NGS excels at larger sample sizes but has lower sensitivity (15%-20% vs. NGS's ~1%).
Video description

This video explores the basics of Sanger sequencing and the fascinating history behind this groundbreaking technology. It explains the basics of DNA sequencing, including the components necessary for the reaction and the steps involved. Topics also include ddNTPs, cycle sequencing, fluorescent dyes and capillary electrophoresis. We'll also compare Sanger sequencing to other sequencing technologies, such as NGS, to give you a better understanding of how it all works. Throughout the video, we'll take a chronological approach to explore the history of Sanger sequencing, starting with its development in the late 1970s and following its evolution. By the end of the video, you'll deeply understand Sanger sequencing and its role in sequencing the human genome. So grab a cup of coffee and journey through the fascinating world of Sanger sequencing! Whether you're a student, researcher, or just curious about the science behind DNA sequencing, this video is for you! 🌏 https://www.clevalab.com 📖 This video is also a blog post with images, references and a PDF download; visit: https://www.clevalab.com/post/sanger-dna-sequencing 👉 You may also like: "Next Generation Sequencing - A Step-By-Step Guide to DNA Sequencing." https://youtu.be/WKAUtJQ69n8 Visit the ClevaLab Channel Page for more videos: https://www.youtube.com/@ClevaLab ✉️ Sign up to the email list to be notified of new videos: https://www.clevalab.com/email-subscription SOCIAL MEDIA LINKS Twitter: https://twitter.com/_ClevaLab Instagram: https://www.instagram.com/_clevalab Pinterest: https://www.pinterest.com.au/ClevaLab Tiktok: https://www.tiktok.com/@clevalab Facebook: https://www.facebook.com/ClevaLab CHAPTERS: 00:00 The 1977 Invention of Sanger Sequencing 00:23 The Basics of How DNA Is Copied 00:36 What are dNTPs & ddNTPs anyway? 01:19 How Does a ddNTP Stop DNA Copying? 01:54 The DNA Naming Convention 5' to 3' 02:43 Which Bases Go Together in Base Pairing? 03:05 Steps of the First Sanger Sequencing Method 06:11 The First DNA Sequencing Instrument, the AB370A. 07:20 The Launch of the Human Genome Project 07:46 How Does Cycle Sequencing Improve Things? 08:59 Automated DNA Separation by Capillary Electrophoresis 09:36 ABI PRISM 310, Modern Sanger Sequencing 10:49 Fluorescent ddNTPs and the Invention of BigDyes 11:33 The Instrument That Sequenced the Genome 13:09 Why Use Sanger Sequencing When There's NGS? #ClevaLab #sequencing #sangersequencing