[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fVbxJpMVdvIt83HwLHshA__BNyS2h7cMYDLjlmUi6n0A":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":47},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":36,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":39,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"body":43,"faq":44,"tags":45,"related":46},"dna-sequencing-sanger-sequencing-method","Sanger Sequencing Method: Principle and Steps",null,"Aastha Shrestha","2022-11-26","2025-12-29",false,"molecular-biology","DNA (deoxyribonucleic acid) sequencing is the process of identifying the exact sequence of nucleotides: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T) in the genome or DNA molecule. In order to determine the sequence, the first DNA sequencing method, the **“chain termination method,” or Sanger sequencing**was developed in 1997 by Frederick Sanger, using radiolabeled partially digested fragments. It enabled Frederick Sanger and his team to sequence the phiX174 virus’s first complete genome.  \n\n**Sanger sequencing is the method of sequencing where incorporation of chain terminating oligonucleotides occurs as primers during in vitroDNA replication.**  [2]\n\n**Single-stranded DNA molecules, DNA polymerase, four deoxyribonucleotide triphosphates (dNTPs; dATP, dCTP, dGTP, and dTTP), and dideoxyribonucleotides triphosphates (ddNTPs; ddATP, ddCTP, ddGTP, and ddTTP) labeled with various fluorescent markers are the essential components for sequencing.**\n\n## Principle of Sanger Sequencing\n\nThe Sanger Sequencing method synthesizes the DNA complementary to a single-stranded DNA template by adding deoxynucleotide triphosphate and dideoxynucleotide triphosphates labeled with a different fluorescent marker that terminates the elongation. Based on these labels, the sequence is identified.\n\n![Process of Sanger sequencing - Process of Sanger Sequencing](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fsanger-sequencing.png)\n\u003Cfigcaption>Figure: Process of Sanger Sequencing\u003C\u002Ffigcaption>\n\n## Components of Sequencing\n\n**DNA template**  \n\nThe starting material for sequencing is a single-stranded DNA template to be sequenced. In the traditional method, single-stranded DNA was obtained using a vector or by denaturing double-stranded DNA with an alkali or boiling method. However, nowadays, [polymerase chain reaction (PCR) method](\u002Fpolymerase-chain-reaction-pcr-steps-types-applications\u002F) helps in obtaining single-stranded DNA.  \n\n**Primer**\n\nPrimer is a short oligonucleotide complementary to a short sequence of DNA templates and anneals with it. The region of the template molecule that will be sequenced is the crucial role that the primer plays.  \n\n**DNA polymerase enzyme**\n\nIt extends the primer and adds nucleotide complementary to the template. This polymerase lacks exonuclease activity as it may degrade the nucleotide from either 5ʹ- 3ʹ or 3ʹ- 5ʹ and affects the accuracy of determining the sequence.  \n\n**Nucleotide triphosphate:** Two different types of nucleotides are used in this method: deoxyribonucleotide triphosphates and dideoxyribonucleotide triphosphates.  \n\n![Deoxynucleotide triphospate - Deoxynucleotide triphospate (Source: Biology 2e, Genetics, Biotechnology and Genomics, Whole Genome Sequencing.)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fdeox.png)\n\u003Cfigcaption>Figure: Deoxynucleotide triphospate (Source: Biology 2e, Genetics, Biotechnology and Genomics, Whole Genome Sequencing.)\u003C\u002Ffigcaption>\n\n1. **Deoxyribonucleotide triphosphates:** These are typical nucleotide triphosphates, which comprise nitrogenous bases (A, T, G, C), ribose sugar with a hydroxyl group at 3ʹ carbon and phosphate group at the 5ʹ carbon respectively, which aids in the formation of a phosphodiester bond between each deoxyribonucleotide triphosphates.\n2. **Dideoxyribonucleotide triphosphates:** These are modified nucleotide triphosphates so that the 3ʹ carbon of the ribose sugar does not have a hydroxyl group. So, these are used as 3ʹ-end chain terminators as the phosphodiester bond does not form with the next incoming nucleotide.\n\n![Dideoxynucleotide triphospate - Dideoxynucleotide triphospate (Source: Brown, TA. Gene Cloning And DNA Analysis An  Introduction)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fddntp-1.png)\n\u003Cfigcaption>Figure: Dideoxynucleotide triphospate (Source: Brown, TA. Gene Cloning And DNA Analysis An  Introduction)\u003C\u002Ffigcaption>\n\n## Steps Involved in Sanger Sequencing\n\n### Elongation and chain termination\n\nThe DNA template to be sequenced is subjected to Polymerase Chain Reaction (PCR), except that dideoxynucleotide triphosphate is also included. dNTPs, DNA polymerase, primer, and template are all in the typical PCR reaction.\n\nThe primer anneals to the template DNA, and DNA polymerase extends the primer by randomly adding dNTPs or ddNTPs, but once the ddNTP is incorporated, the reaction gets terminated. These deoxynucleotides are larger than dideoxynucleotides, so the termination does not occur near the primer region. For example, if dATP binds during the elongation, it ceases the elongation, and the sequence is read as A (shown below). Similarly, if ddGTP, ddTTP, or ddCTP is added, the sequence is read as G, T, or C, respectively.\n\n![Annealing of primer to the template DNA  - Annealing of primer to the template DNA (Source: Brown, TA. Gene Cloning And DNA  Analysis An Introduction)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fini.png)\n\u003Cfigcaption>Figure: Annealing of primer to the template DNA (Source: Brown, TA. Gene Cloning And DNA  Analysis An Introduction)\u003C\u002Ffigcaption>\n\n![ Elongation of DNA molecule and termination after the addition of ddATP  - Elongation of DNA molecule and termination after the addition of ddATP (Source:  Brown, TA. Gene Cloning And DNA Analysis An Introduction)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fteminate.png)\n\u003Cfigcaption>Figure: Elongation of DNA molecule and termination after the addition of ddATP (Source:  Brown, TA. Gene Cloning And DNA Analysis An Introduction)\u003C\u002Ffigcaption>\n\n**The traditional method performs these reactions** in four tubes containing typical PCR mixtures. Still, each tube includes either dideoxynucleotide triphosphate (ddATP, ddCTP, ddTTP, or ddGTP) that is radioactively labeled.  \n\n**On the other hand,** nowadays, all these reactions are performed in a single tube with ddNTPs, each labeled with a different fluorescent dye.  \n\n### Electrophoresis and Sequence Identification\n\nAfter the amplification,[electrophoresis](\u002Felectrophoresis-principles-types-and-uses\u002F) is carried out. The mixture is loaded either in the polyacrylamide gel (in the traditional method) or into a capillary gel system tube. These molecules are separated according to their lengths; each contains dideoxynucleotide at their end.  \n\n**In the traditional method,** the products are separated through polyacrylamide gel gel electrophoresis in four separate lanes and are scored according to their molecular masses, as shown below. Based on their masses, and the radiolabelled ddNTPs, the bases are identified. Their sequences are shown on the left side of the figure below. The identification is done with the help of the [spectrophotometer](\u002Fspectrophotometer-principle-parts-types-uses\u002F).\n\n![Gel electrophoresis of radioactive labelled product in four different lanes - Gel electrophoresis of radioactive labelled product in four different lanes (Source: Janitz, M. (2008). Next-Generation Genome Sequencing.)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FTra1.png)\n\u003Cfigcaption>Figure: Gel electrophoresis of radioactive labelled product in four different lanes (Source: Janitz, M. (2008). Next-Generation Genome Sequencing.)\u003C\u002Ffigcaption>\n\n**In the new technique,** the products are separated in a single glass capillary filled with a polymer and are passed through a fluorescent detector that determines if each molecule ends in A, T, G, or C based on the label attached to the dideoxynucleotides.\n\n![Detection of each sequences of DNA passed through the detector  - Detection of each sequences of DNA passed through the detector (Source: Brown, TA.  (2010). Gene Cloning And DNA Analysis An Introduction.)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fdetection.png)\n\u003Cfigcaption>Figure: Detection of each sequences of DNA passed through the detector (Source: Brown, TA.  (2010). Gene Cloning And DNA Analysis An Introduction.)\u003C\u002Ffigcaption>\n\n![DNA sequences in a graph form detector  - DNA sequences in a graph form detector (Source: Brown, TA. (2010). Gene Cloning And  DNA Analysis An Introduction. )](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fgraph.png)\n\u003Cfigcaption>Figure: DNA sequences in a graph form detector (Source: Brown, TA. (2010). Gene Cloning And  DNA Analysis An Introduction. )\u003C\u002Ffigcaption>\n\n## Advantages of Sanger Sequencing\n\nCompared to other methods of sequencing, Sanger sequencing have many advantages which are as follows:\n\n- It is more specific for testing the variants of the same families.\n- Extensive validation is not required as the method is verified on one or a small number of sequences of interest.  \n- Less reliant on computational tools.\n- Cost-effective for a single sample.  \n\n## Limitations of Sanger Sequencing\n\nAlthough the Sanger sequencing is the preferable method of DNA sequencing it has some limitations, which are as follows:\n\n- It only sequences short fragments of DNA of about 300-1kb bases.\n- Cannot detect different genes simultaneously.\n- It requires a more significant amount of DNA as an input.\n- As primer binds to the first 15 – 40 bases, the quality of sequences in this region is often poor.\n- It is a time-consuming method.  \n- If the traditional method is used, the cloning vector sequences may be present in the final sequences.\n\n**REFERENCES**\n\n1. Brown, T. (2010). *Gene Cloning And DNA Analysis An Introduction*. A John Wiley & Sons,  Ltd., Publication.\n2. Clark, D. P., Pazdernik, N. J., & McGehee, M. R. (2010). In *Molecular biology: Academic cell update* (Third, pp. 241–242). essay, Academic Press\u002FElsevier.\n3. Janitz, M. (2008). *Next-Generation Genome Sequencing*. WILEY-VCH Verlag GmbH & Co.  KGaA.  \n4. \u003Chttps:\u002F\u002Fthe-dna-universe.com\u002F2020\u002F11\u002F02\u002Fa-journey-through-the-history-of-dna-sequencing\u002F>  \n5. Timeline: History of genomics. . @Yourgenome · Science Website.  [https:\u002F\u002Fwww.yourgenome.org\u002Ffacts\u002Ftimeline-history-of-genomics\u002F](https:\u002F\u002Fwww.yourgenome.org\u002Ffacts\u002Ftimeline-history-of-genomics\u002F)\n6. \u003Chttps:\u002F\u002Fwww.news-medical.net\u002Flife-sciences\u002FChallenges-with-Sanger-Sequencing.aspx>\n7. \u003Chttps:\u002F\u002Fwww.mlo-online.com\u002Fhome\u002Farticle\u002F13009097\u002Fback-to-basics-sanger-sequencing-and> its-applications",[],[],[],[48,55,62,67,71,75,79,84,88,92],{"slug":49,"name":50,"description":51,"image":52,"body":53,"postCount":54},"acharya-tankeshwar","Acharya Tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":56,"name":57,"description":58,"image":59,"body":60,"postCount":61},"ashma-shrestha","Ashma Shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":63,"name":64,"description":65,"image":37,"body":37,"postCount":66},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":68,"name":69,"description":65,"image":37,"body":37,"postCount":70},"samikshya-acharya","Samikshya Acharya",20,{"slug":72,"name":73,"description":65,"image":37,"body":37,"postCount":74},"alisha-tripathi","Alisha Tripathi",6,{"slug":76,"name":38,"description":77,"image":37,"body":37,"postCount":78},"aastha-shrestha"," Author \u002F Contributor",9,{"slug":80,"name":81,"description":82,"image":37,"body":37,"postCount":83},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":85,"name":86,"description":65,"image":37,"body":37,"postCount":87},"srijana-khanal","Srijana Khanal",18,{"slug":89,"name":90,"description":82,"image":37,"body":37,"postCount":91},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":93,"name":94,"description":65,"image":37,"body":95,"postCount":96},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]