Sanger Sequencing Principles
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Questions and Answers

What is the primary function of dideoxynucleotides in Sanger sequencing?

  • To initiate DNA synthesis
  • To extend DNA synthesis
  • To purify the target DNA
  • To terminate DNA synthesis (correct)

What is the purpose of primer annealing in Sanger sequencing?

  • To bind the primer to the target DNA (correct)
  • To separate the fragments by size
  • To terminate DNA synthesis
  • To extend the primer

What is the advantage of Sanger sequencing in terms of DNA fragment length?

  • It can sequence long DNA fragments up to 100 bp
  • It can only sequence RNA fragments
  • It can only sequence short DNA fragments
  • It can sequence long DNA fragments up to 1,000 bp (correct)

What is a limitation of Sanger sequencing?

<p>It is limited to sequencing short to medium-length DNA fragments (A)</p> Signup and view all the answers

What is the purpose of gel electrophoresis in Sanger sequencing?

<p>To separate the fragments by size (B)</p> Signup and view all the answers

What is an advantage of Sanger sequencing in terms of accuracy?

<p>It has high accuracy and reliability (C)</p> Signup and view all the answers

What is the purpose of the fluorescent dyes in Sanger sequencing?

<p>To detect and identify the fragments (A)</p> Signup and view all the answers

What is a disadvantage of Sanger sequencing in terms of time?

<p>It is a time-consuming and labor-intensive process (D)</p> Signup and view all the answers

Study Notes

Sanger Sequencing

Principle

  • Uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis, resulting in a series of fragments of varying lengths
  • Each ddNTP is labeled with a different fluorescent dye, allowing for detection and identification

Procedure

  1. Template preparation
    • Isolate and purify the target DNA
    • Prepare a single-stranded DNA template
  2. Primer annealing
    • Design and synthesize a primer that binds to the target DNA
    • Anneal the primer to the template DNA
  3. Extension and termination
    • Add dNTPs and ddNTPs to the reaction mixture
    • DNA polymerase extends the primer, incorporating dNTPs and ddNTPs
    • ddNTPs terminate the reaction, resulting in a series of fragments
  4. Gel electrophoresis
    • Separate the fragments by size using gel electrophoresis
    • The different fluorescent dyes allow for detection and identification of the fragments
  5. Sequencing
    • Analyze the resulting gel or chromatogram to determine the DNA sequence

Advantages

  • High accuracy and reliability
  • Can sequence long DNA fragments (up to 1,000 bp)
  • Can be used for both DNA and RNA sequencing

Limitations

  • Time-consuming and labor-intensive
  • Limited to sequencing short to medium-length DNA fragments
  • Requires a large amount of high-quality DNA template

Sanger Sequencing

Principle

  • Dideoxynucleotides (ddNTPs) are used to terminate DNA synthesis, resulting in a series of fragments of varying lengths
  • Each ddNTP is labeled with a different fluorescent dye, allowing for detection and identification

Procedure

Template Preparation

  • Isolate and purify the target DNA
  • Prepare a single-stranded DNA template

Primer Annealing

  • Design and synthesize a primer that binds to the target DNA
  • Anneal the primer to the template DNA

Extension and Termination

  • Add dNTPs and ddNTPs to the reaction mixture
  • DNA polymerase extends the primer, incorporating dNTPs and ddNTPs
  • ddNTPs terminate the reaction, resulting in a series of fragments

Gel Electrophoresis

  • Separate the fragments by size using gel electrophoresis
  • Different fluorescent dyes allow for detection and identification of the fragments

Sequencing

  • Analyze the resulting gel or chromatogram to determine the DNA sequence

Advantages

  • High accuracy and reliability
  • Can sequence long DNA fragments (up to 1,000 bp)
  • Can be used for both DNA and RNA sequencing

Limitations

  • Time-consuming and labor-intensive
  • Limited to sequencing short to medium-length DNA fragments
  • Requires a large amount of high-quality DNA template

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Description

Learn about the principles and procedures of Sanger sequencing, a method used to determine the order of nucleotides in DNA. Quiz covers the use of dideoxynucleotides, primer annealing, and other key concepts.

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