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    what is the difference between pcr and sanger sequencing with regard to the materials needed to perform these reactions?

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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).

    Source : www.sigmaaldrich.com

    DNA sequencing (article)

    How the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA is determined.

    Biotechnology

    DNA sequencing

    How the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA is determined.

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    Key points:

    DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.

    In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be determined.

    Next-generation sequencing techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing.

    What is sequencing?

    You may have heard of genomes being sequenced. For instance, the human genome was completed in 2003, after a many-year, international effort. But what does it mean to sequence a genome, or even a small fragment of DNA?

    DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward.

    Sequencing an entire genome (all of an organism’s DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus." However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project

    ^1 1

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    In this article, we’ll take a look at methods used for DNA sequencing. We'll focus on one well-established method, Sanger sequencing, but we'll also discuss new ("next-generation") methods that have reduced the cost and accelerated the speed of large-scale sequencing.

    Sanger sequencing: The chain termination method

    Regions of DNA up to about

    900 900 900

    base pairs in length are routinely sequenced using a method called Sanger sequencing or the chain termination method. Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977.

    In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. (These fragments weren't necessarily

    900 900 900

    bp or less, but researchers were able to "walk" along each fragment using multiple rounds of Sanger sequencing.) The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.

    Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).

    Ingredients for Sanger sequencing

    Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies DNA in vitro. They include:

    A DNA polymerase enzyme

    A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase

    The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)

    The template DNA to be sequenced

    However, a Sanger sequencing reaction also contains a unique ingredient:

    Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye

    _Image credit: "Whole-genome sequencing: Figure 1," by OpenStax College, Biology (CC BY 4.0)._

    Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a new nucleotide to be added to an existing chain.

    Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available and no further nucleotides can be added. The chain ends with the dideoxy nucleotide, which is marked with a particular color of dye depending on the base (A, T, C or G) that it carries.

    [Where is the dye attached?]

    Method of Sanger sequencing

    The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.

    The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.

    Source : www.khanacademy.org

    Sanger Sequencing

    Sanger Sequencing

    Sanger sequencing is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication;

    From: Genomics, Circuits, and Pathways in Clinical Neuropsychiatry, 2016

    Related terms:

    Single-Nucleotide PolymorphismExonPyrosequencingPlasmidsPolymerase Chain ReactionPhenotypeDNARNA

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    Somatic Mosaicism and Neurological Diseases

    Saumya S. Jamuar, ... Christopher A. Walsh, in Genomics, Circuits, and Pathways in Clinical Neuropsychiatry, 2016

    Sanger Sequencing

    Sanger sequencing is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication; it is the most widely used method for the detection of SNVs. Because both alleles of an autosomal locus are sequenced concurrently and are displayed as an analogue electropherograms, Sanger sequencing is unable to detect mosaic alleles below a threshold of 15–20% (Rohlin et al., 2009) and can miss a significant proportion of low-level mosaic mutations (Jamuar et al., 2014). In addition, mosaic mutations at higher allele fractions are miscalled “germ line,” which highlights the limitations of Sanger sequencing in detecting mosaicism on both ends of the spectrum (Jamuar et al., 2014).

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    Overview of Technical Aspects and Chemistries of Next-Generation Sequencing

    Ian S. Hagemann, in Clinical Genomics, 2015

    Applications in Clinical Genomics

    Sanger sequencing is a “first-generation” DNA sequencing method. Despite the advantages of next-generation sequencing techniques, where throughput is orders of magnitude higher, Sanger sequencing retains an essential place in clinical genomics for at least two specific purposes.

    First, Sanger sequencing serves as an orthogonal method for confirming sequence variants identified by NGS. When validating clinical NGS tests, reference materials sequenced by Sanger approaches provide ground truth against which the NGS assay can be benchmarked. These materials may include well-characterized publicly available reagents, such as cell lines studied in the HapMap project, or archival clinical samples previously tested by Sanger methods.

    As an orthogonal method, Sanger sequencing provides a means to confirm variants identified by NGS. It would be impractical to Sanger-confirm every variant, given the large number of primers, reactions, and interpretations that would be required. However, there may be instances where the veracity of a specific variant is in doubt; e.g., called variants that are biologically implausible or otherwise suspected of being spurious. Sanger sequencing is the easiest method to resolve these uncertainties and is therefore an invaluable protocol in any clinical genomics laboratory.

    Second, Sanger sequencing provides a means to “patch” the coverage of regions that are poorly covered by NGS. In targeted NGS testing, there may be regions that are resistant to sequencing, due to poor capture, amplification, or other idiosyncrasies. These regions are often rich in GC content. One approach to restoring coverage of these areas is to increase the quantity of input DNA, but the quantity available may be limited. It may be possible to redesign the amplification step or capture reagents, or otherwise troubleshoot the NGS technology. However, a very practical approach, when the area to be backfilled is small, is to use Sanger sequencing to span the regions poorly covered by NGS.

    When Sanger sequencing is used for backfilling NGS data, the NGS and Sanger data must be integrated together for purposes of analysis and reporting, which represents a challenge since these data are obtained by different methods and do not have a one-to-one correspondence to one another. Analyses that are natural for NGS data may be difficult to map onto data obtained by Sanger. For example, measures of sequence quality that are meaningful for NGS are not applicable to Sanger; the concept of depth of coverage can only be indirectly applied to Sanger data; allele frequencies are indirectly and imprecisely ascertained in Sanger sequence from peak heights rather than read counts; and Sanger data do not have paired ends. While NGS may potentially be validated to allow meaningful variant calling from a single nonreference read, the sensitivity of Sanger sequencing has a floor of approximately 20%: variants with a lower allele frequency may be indistinguishable from noise or sequencing errors (discussed below). Thus the performance of an NGS assay may be altered in areas of Sanger patching, and these deviations in performance must be documented and/or disclaimed.

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    Genetic Testing Techniques

    Alicia Gomes MS, Bruce Korf MD, PhD, in Pediatric Cancer Genetics, 2018

    Sanger Sequencing

    Methodology

    Sanger sequencing is a targeted sequencing technique that uses oligonucleotide primers to seek out specific DNA regions. Sanger sequencing begins with denaturation of the double-stranded DNA. The single-stranded DNA is then annealed to oligonucleotide primers and elongated using a mixture of deoxynucleotide triphosphates (dNTPs), which provide the needed arginine (A), cytosine (C), tyrosine (T), and guanine (G) nucleotides to build the new double-stranded structure. In addition, a small quantity of chain-terminating dideoxynucleotide triphosphates (ddNTPs) for each nucleotide is included. The sequence will continue to extend with dNTPs until a ddNTP attaches. As the dNTPs and ddNTPs have an equal chance of attaching to the sequence, each sequence will terminate at varying lengths.

    Source : www.sciencedirect.com

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