DNA Sequencing: Sanger Method Overview
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Questions and Answers

What is the primary purpose of DNA sequencing?

  • To determine the order of bases in DNA (correct)
  • To identify different types of RNA
  • To isolate DNA from bacterial cells
  • To amplify DNA through PCR

    • Which of the following best describes the Sanger sequencing method?

  • It uses multiple primers for DNA synthesis.
  • It requires a complex array of enzymes to function.
  • It is classified as a first generation sequencing method. (correct)
  • It is a second generation sequencing technique.

    • What are ddNTPs in the context of Sanger sequencing?

  • Nucleotides that lack phosphate groups
  • Standard nucleotides used in PCR amplification
  • Nucleotides that promote continuous DNA synthesis
  • Fluorescently labeled nucleotides that cause chain termination (correct)

    • What role does the single primer play in the Sanger sequencing process?

    <p>It initiates DNA synthesis from the 3’ end of the template.</p> Signup and view all the answers

    Which advancement in genetic research has DNA sequencing contributed to significantly?

    <p>The identification of mutations associated with diseases</p> Signup and view all the answers

    What is the first step of the Sanger sequencing method?

    <p>DNA denaturation and template preparation</p> Signup and view all the answers

    Who developed the Sanger sequencing method?

    <p>Frederick Sanger and his colleagues</p> Signup and view all the answers

    What is a significant difference between dNTPs and ddNTPs?

    <p>ddNTPs lack the 3’ hydroxyl group required for DNA extension.</p> Signup and view all the answers

    What is the role of labelled dideoxynucleotides (ddNTPs) in the Sanger sequencing method?

    <p>To terminate the growing DNA chain</p> Signup and view all the answers

    Which of the following statements describes an outcome of capillary electrophoresis in Sanger sequencing?

    <p>Each amplicon is separated based on its length.</p> Signup and view all the answers

    What components are included in the mixture for preparing template DNA in Sanger sequencing?

    <p>A mixture of dNTPs and ddNTPs</p> Signup and view all the answers

    Why are smaller amounts of ddNTPs used compared to dNTPs in the sequencing process?

    <p>To ensure that chain termination occurs at various lengths</p> Signup and view all the answers

    What is the primary function of the fluorescent tag on the dideoxynucleotides in Sanger sequencing?

    <p>To generate a chromatogram</p> Signup and view all the answers

    During the Sanger sequencing process, what directly excites the labels on the nucleotides?

    <p>A laser</p> Signup and view all the answers

    What is the result seen in the chromatogram generated by Sanger sequencing?

    <p>A sequence of peaks corresponding to different nucleotides</p> Signup and view all the answers

    In Sanger sequencing, what happens once a dideoxynucleotide is incorporated into the growing DNA chain?

    <p>The chain is terminated and no further bases can be added</p> Signup and view all the answers

    What characterized the Human Genome Project?

    <p>It was completed in 2003 using Sanger sequencing.</p> Signup and view all the answers

    Next Generation Sequencing was first introduced in 2000.

    <p>False</p> Signup and view all the answers

    What is one major benefit of Next Generation Sequencing compared to first-generation methods?

    <p>Increased speed and accuracy</p> Signup and view all the answers

    Next Generation Sequencing is sometimes referred to as __________ sequencing.

    <p>second-generation</p> Signup and view all the answers

    Match the following NGS categories with their descriptions:

    <p>Sequencing by hybridization = Uses specific probes to interrogate sequences Sequencing by Synthesis = Further development of Sanger sequencing RNA sequencing (RNA-seq) = Quantify mRNAs for gene expression analysis Deep sequencing = Rapidly sequence whole genomes</p> Signup and view all the answers

    Which of the following is an application of NGS?

    <p>Analysing epigenetic factors</p> Signup and view all the answers

    Next Generation Sequencing allows the identification of novel RNA variants.

    <p>True</p> Signup and view all the answers

    What is the purpose of adapter sequences in library preparation?

    <p>To provide a universal priming site for sequencing primers</p> Signup and view all the answers

    Sequencing machines amplify each library fragment on a solid surface, such as beads or flat silicon.

    <p>True</p> Signup and view all the answers

    What is a common characteristic of Sequencing by Synthesis (SBS)?

    <p>Relies on shorter reads and has higher error rates compared to Sanger sequencing.</p> Signup and view all the answers

    The raw data provided by sequencing machines consists of a collection of _____________ generated at each cluster.

    <p>DNA sequences</p> Signup and view all the answers

    Match the following properties with their relevance in sequencing:

    <p>Shorter reads (300-500 bp) = Sequencing by Synthesis (SBS) Higher error rate = Sequencing by Synthesis (SBS) Massively parallel sequencing = High sequence coverage Reversibly fluorescent nucleotides = Step-by-step nucleotide incorporation</p> Signup and view all the answers

    What happens to each cluster during the sequencing process?

    <p>Each cluster acts as an individual sequencing reaction</p> Signup and view all the answers

    Sequencing by Synthesis uses only one type of nucleotide for DNA sequencing.

    <p>False</p> Signup and view all the answers

    What is the significance of high sequence coverage in Sequencing by Synthesis?

    <p>It allows for the generation of millions to billions of short DNA sequence reads.</p> Signup and view all the answers

    Sequencing by Synthesis relies on _____________ incorporation of nucleotides.

    <p>step-by-step</p> Signup and view all the answers

    What is the primary function of reversible termination in nucleotide sequencing?

    <p>To ensure only one nucleotide is incorporated per cycle</p> Signup and view all the answers

    In Illumina sequencing, PCR amplification is performed after the library preparation step.

    <p>True</p> Signup and view all the answers

    What is the typical length of DNA fragments after fragmentation during library preparation?

    <p>200 – 600 bp</p> Signup and view all the answers

    The process of ________ occurs to bind complementary DNA to primers on the surface of the flow cell.

    <p>washing</p> Signup and view all the answers

    Match the following NGS technologies with their characteristics:

    <p>454 sequencing = Utilizes pyrosequencing Illumina = Cyclic reversible termination SOLiD = Uses ligation-based sequencing Ion Torrent = Measures pH changes during sequencing</p> Signup and view all the answers

    During bridge amplification in Illumina sequencing, what is the main purpose of the flexible linker?

    <p>To facilitate contact between the DNA template and primers</p> Signup and view all the answers

    In the sequencing process, fluorescent signals are read only after the clustering of DNA fragments.

    <p>True</p> Signup and view all the answers

    What type of molecules are cleaved and washed away after the fluorescent signal is read?

    <p>Fluorescent molecules and terminator groups</p> Signup and view all the answers

    The first step in the Illumina sequencing process is ________ preparation.

    <p>library</p> Signup and view all the answers

    Study Notes

    DNA Sequencing

    • DNA sequencing determines the order of bases in a DNA sequence.
    • It has aided in our understanding of genetics, with applications such as tackling human diseases and studying evolution.

    Sanger Sequencing Method

    • Also known as the "chain termination method."
    • A first-generation sequencing method.
    • Developed in 1977 by Frederick Sanger and his colleagues (who later sequenced an entire virus genome).
    • Uses high fidelity DNA polymerase to create a complementary copy from a single-stranded DNA template.
    • Requires a single primer complementary to the template to initiate DNA synthesis from the 3' end.

    Sanger Sequencing: Chain Termination Method

    • Uses ddNTPs (dideoxynucleotides) which resemble DNA monomers and are incorporated into the growing chain.
    • ddNTPs differ from dNTPs in two ways:
      • They lack a 3' hydroxyl group needed for further DNA extension, causing chain termination upon incorporation.
      • Each ddNTP is tagged with a fluorescent dye, allowing for automatic detection.

    Components for Sanger Sequencing

    • DNA template
    • Primer
    • dNTPs (dATP, dTTP, dCTP, dGTP)
    • ddNTPs (ddATP, ddTTP, ddCTP, ddGTP)
    • DNA polymerase

    Sanger Sequencing Method: Steps

    • Step 1: The DNA to be sequenced is amplified using PCR, and the double helix is denatured with heat to separate the strands.
    • Step 2: A mixture of dNTPs (normal nucleotides), ddNTPs (labeled dideoxynucleotides), DNA primer, and DNA polymerase is added to the template.
    • Step 3: The polymerase creates a copy of the DNA template, but the process is terminated when a ddNTP is incorporated, resulting in fragments of varying lengths.
    • Step 4: Fragments are separated by capillary electrophoresis, where shorter fragments move faster due to their smaller size. The laser excites the fluorescent dye on each ddNTP, emitting light for detection. This creates a chromatogram that shows the fluorescent peaks of each nucleotide, indicating the sequence from shortest to longest.

    Human Genome Project

    • The first major effort in DNA sequencing was the Human Genome Project.
    • This project used Sanger Sequencing (chain termination method)
    • The project took 13 years to complete.
    • The project cost $3 billion.
    • The project was completed in 2003.

    Next Generation Sequencing (NGS)

    • NGS is a newer technology.
    • Second-generation sequencing was introduced in 2004 and 2006.
    • Offers high-throughput sequencing.

    Applications of NGS

    • NGS can be used to sequence the entire genome.
    • NGS can be used to sequence specific regions of DNA or RNA.
    • NGS has revolutionized the life sciences.
    • Used to discover novel RNA variants and splice sites.
    • Used to quantify mRNAs for gene expression analysis.
    • Used to study epigenetic factors.
    • Used to study cancer samples by studying rare somatic variants and tumor subclones.
    • Used to identify novel pathogens.

    Categories of NGS

    • Sequencing by hybridization: uses specific probes to identify sequences.
      • Used for diagnostic purposes, including:
        • identifying disease-related SNPs.
        • identifying gross chromosome abnormalities, such as rearrangements, deletions, duplications, and copy number variants (CNVs).
    • Sequencing by Synthesis (SBS): an improvement on Sanger Sequencing that does not use ddNTPs.
      • Uses repeated synthesis cycles and methods to incorporate nucleotides into a growing chain.
      • There are other methods like Pyrosequencing but are not discussed here.

    Similarities Between Different NGS Technologies

    • Sample preparation:
      • Requires a library obtained by amplification or ligation with custom adapter sequences.
      • Adapter sequences allow library hybridization to the sequencing chips.
        • These sequences provide a universal priming site for sequencing primers.
    • Sequencing Machines:
      • Each library fragment is amplified on a solid surface – either beads or a flat silicon-derived surface.
      • This is done using covalently attached DNA linkers, which hybridize the library adapters.
      • Amplification creates clusters of DNA, each originating from a single library fragment.
        • Each cluster acts as an individual sequencing reaction.
        • The sequence from each cluster is optically read, either by light or fluorescence.
        • Each machine has its cycle conditions.
    • Data Output:
      • Each machine provides raw data at the end of the sequencing run.
      • Raw data is a collection of DNA sequences generated at each cluster.
      • This data is further analyzed to provide meaningful results.

    Sequencing by Synthesis (SBS) Properties

    • Relies on shorter reads (300-500 bp).
    • Generally, it has a higher error rate than Sanger sequencing.
      • Due to incomplete removal of the fluorescent signal, which can cause higher background noise levels.
    • Relies on high sequence coverage, which is referred to as “massively parallel sequencing”.
      • Of millions to billions of short DNA sequence reads (50 – 300 nucleotides)
      • This is called Short Read Sequencing.
    • Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides.
      • Nucleotides are modified in two ways:
        • Each nucleotide is reversibly attached to a single fluorescent molecule with unique emission wavelengths.
        • Each nucleotide is reversibly terminated, ensuring only one nucleotide is incorporated per cycle.

    Sequencing by Synthesis (SBS) Process

    • All four nucleotides are added to the sequencing chip.
    • A single nucleotide is incorporated into the sequence.
    • Remaining nucleotides are washed away.
    • The fluorescent signal is read at each cluster and recorded.
    • Both the fluorescent molecule and the terminator group are cleaved and washed away.
    • This process is repeated until sequencing is complete.

    Illumina Sequencing (Solexa Sequencing) - Cyclic Reversible Termination

    • Step 1: Library Preparation

      • DNA is broken up into more manageable fragments (~200 – 600 bp).
      • Short sequences of DNA (adapters) are attached to the DNA fragments.
      • The DNA fragments attached to the adapters are denatured (made single-stranded).
      • This process constructs a mix of adapter-flanked fragments up to several hundred bp in length.

    • Step 2: Bridge Amplification

      • Illumina technology relies on bridge PCR to amplify the genomic region being sequenced.
      • An in vitro constructed adapter-flanked library is PCR amplified.
        • Both primers densely coat the surface of a solid substrate, attached at their 5’ end by a flexible linker.
        • Due to this, amplification products from the library remain locally attached near the point of origin.

    • Step 3: Sequencing Reaction

      • Components:

        • Primers
        • dNTPs: labelled with a fluorescent dye and contain a reversible terminator (Trinitrogen = N3)
        • DNA polymerase

      • Process:

        • Cyclic reversible termination
        • Only one of four fluorescent dNTPs is added per cycle.
        • Images of clusters are captured after the incorporation of each nucleotide.
        • After imaging, the fluorescent dye and terminator are cleaved and released.

    When to Use NGS vs Sanger Sequencing

    • Sanger Sequencing:
      • A good choice when investigating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer).
    • NGS:
      • Allows for cost-effective screening of more samples.
      • Allows for the detection of multiple variants across targeted areas of the genome.
      • Used when Sanger sequencing is too costly and time-consuming.

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    Related Documents

    DNA Sequencing Lecture Notes PDF
    DNA Sequencing: Next Generation Sequencing (NGS) Lecture PDF

    Description

    Explore the Sanger sequencing method, known for its chain termination technique. This quiz covers the fundamentals of DNA sequencing, its significance in genetics, and the use of ddNTPs in the process. Test your knowledge on this pivotal advancement in molecular biology.

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