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    Sanger Sequencing Steps & Method

    Learn about Sanger Sequencing steps or the chain termination method and how DNA sequencing works and how to read Sanger Sequencing results accurately for your research.

    Sanger Sequencing Steps & Method

    Sanger Sequencing Steps & Method

    Sanger Sequencing Steps & Method What is Sanger Sequencing?

    Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA. The method was developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the name the Sanger Sequence.

    To review the general structure of DNA, please see Figure 2.

    How Does Sanger Sequencing Work?

    Sanger sequencing can be performed manually or, more commonly, in an automated fashion via sequencing machine (Figure 1). Each method follows three basic steps, as described below.

    Figure 1.Three Basic Steps of Automated Sanger Sequencing.

    Sanger Sequencing Steps

    There are three main steps to Sanger sequencing.

    1. DNA Sequence For Chain Termination PCR

    The DNA sequence of interest is used as a template for a special type of PCR called chain-termination PCR. Chain-termination PCR works just like standard PCR, but with one major difference: the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs). In the extension step of standard PCR, DNA polymerase adds dNTPs to a growing DNA strand by catalyzing the formation of a phosphodiester bond between the free 3’-OH group of the last nucleotide and the 5’-phosphate of the next (Figure 2).

    In chain-termination PCR, the user mixes a low ratio of chain-terminating ddNTPs in with the normal dNTPs in the PCR reaction. ddNTPs lack the 3'-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases. The result of chain-termination PCR is millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at a random lengths (n) by 5’-ddNTPs.

    In manual Sanger sequencing, four PCR reactions are set up, each with only a single type of ddNTP (ddATP, ddTTP, ddGTP, and ddCTP) mixed in.

    In automated Sanger sequencing, all ddNTPs are mixed in a single reaction, and each of the four dNTPs has a unique fluorescent label.

    2. Size Separation by Gel Electrophoresis

    In the second step, the chain-terminated oligonucleotides are separated by size via gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size. The smaller a fragment is, the less friction it will experience as it moves through the gel, and the faster it will move. In result, the oligonucleotides will be arranged from smallest to largest, reading the gel from bottom to top.

    In manual Sanger sequencing, the oligonucleotides from each of the four PCR reactions are run in four separate lanes of a gel. This allows the user to know which oligonucleotides correspond to each ddNTP.

    In automated Sanger sequencing, all oligonucleotides are run in a single capillary gel electrophoresis within the sequencing machine.

    3. Gel Analysis & Determination of DNA Sequence

    The last step simply involves reading the gel to determine the sequence of the input DNA. Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence (e.g., the shortest fragment must terminate at the first nucleotide from the 5’ end, the second-shortest fragment must terminate at the second nucleotide from the 5’ end, etc.) Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.

    In manual Sanger sequencing, the user reads all four lanes of the gel at once, moving bottom to top, using the lane to determine the identity of the terminal ddNTP for each band. For example, if the bottom band is found in the column corresponding to ddGTP, then the smallest PCR fragment terminates with ddGTP, and the first nucleotide from the 5’ end of the original sequence has a guanine (G) base.

    In automated Sanger sequencing, a computer reads each band of the capillary gel, in order, using fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted. Because each of the four ddNTPs is tagged with a different fluorescent label, the light emitted can be directly tied to the identity of the terminal ddNTP. The output is called a chromatogram, which shows the fluorescent peak of each nucleotide along the length of the template DNA.

    Figure 2.DNA Structure Schematic. DNA is a molecule composed of two strands that coil around each other to form a double helix. Each strand is made up of a string of molecules called deoxyribonucleotides (dNTPs).

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    How Does Sanger Sequencing Work?

    Let’s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it – we’re talking about Sanger Sequencing by capillary electrophoresis. Many might ask, “why is it called Sanger Sequencing?” Sanger Sequencing is named after the inventor of this ground breaking technology, Dr. Frederick Sanger, who

    How Does Sanger Sequencing Work?

    By Jeremy Schoales 06.17.2015

    Let’s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it – we’re talking about Sanger Sequencing by capillary electrophoresis. Many might ask, “why is it called Sanger Sequencing?” Sanger Sequencing is named after the inventor of this ground breaking technology, Dr. Frederick Sanger, who developed this method over 40 years ago in the mid-70s. So, what are the basics of Sanger Sequencing?

    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′ end.

    The extension products are then separated by Capillary Electrophoresis or 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.

    And that, in a nutshell is Sanger Sequencing.

    If you want to learn more, just download our free Sanger Sequencing Handbook.

    We’re making new Seq It Out videos all the time, so if you have an idea the would make a great topic, just drop us a line.

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