Sanger sequencing method

• How does the Sanger method prevent further elongation during DNA replication?

  By incorporating special nucleotides
• How does the DNA replication process work to determine the DNA sequence?

  • The DNA strand to be sequenced is primed
  • Four separate reactions are prepared, each with a different replication-stopping nucleotide (A, T, C, G)
  • The replication products from the 'C' reaction are separated by electrophoresis
  • The sequence is read as complementary bands containing the labeled strands
• What is a dideoxynucleotide (ddNTP)?

  A modified nucleotide lacking the 3'OH group
• Why does a dideoxynucleotide terminate DNA synthesis?

  It lacks the 3'OH group for bonding
• What are ddNTPs used for in the Sanger method?

  They are DNA-chain-stopping nucleotides
• What is the purpose of the fluorescent labeling step in Sanger sequencing?

  To allow detection of the DNA fragments during electrophoresis
• What is the purpose of primer annealing and chain extension in Sanger sequencing?

  • Primers anneal to the template DNA
  • Polymerase extends the DNA chain
  • This creates DNA fragments of varying lengths
• How are the special chain-terminating nucleotides labeled in Sanger sequencing?

  With different fluorescent tags
• What is the temperature of the primer annealing step?

  55°C
• What role does the electric field play in gel electrophoresis?

  It pulls DNA fragments through the gel
• What are the steps involved in the DNA replication process?

  1. Prepare four reaction mixtures, each with a different replication-stopping nucleotide
  2. Primer is added to initiate DNA replication
  3. Replication products of 'C' reaction are separated by electrophoresis
  4. Read sequence as complementary bands containing labeled strands
• What is the temperature of the primer extension step?

  72°C
• What is the first component needed for Sanger sequencing?

  A template DNA strand
• What is the purpose of the primer in the DNA replication process?

  To initiate DNA replication
• What are the special nucleotides used in Sanger sequencing called?

  Chain-terminating nucleotides (ddNTPs)
• What are the regular DNA nucleotides used in Sanger sequencing?

  ATP, TTP, GTP, CTP
• What is the temperature of the denaturation step?

  96°C
• How does the DNA replication process differ from the DNA sequencing process described in the image?

  DNA replication makes copies of the entire DNA strand, while DNA sequencing determines the specific order of nucleotides in a DNA strand
• What are the key steps in the Sanger sequencing process?

  1. Primer annealing and chain extension
  2. ddNTP binding and chain termination
  3. Fluorescent labeling of DNA fragments
  4. Capillary gel electrophoresis and detection
  5. Sequence analysis and reconstruction
• How many times is the thermal cycling process repeated in this procedure?

  The thermal cycling process is repeated 25-35 times
• What is the result after 1 complete cycle of the thermal cycling process?

  • The number of DNA molecules is doubled
• What is the purpose of the primer extension step?

  • To synthesize new DNA strands complementary to the original DNA templates
• How does the ddNTP binding and chain termination step work in Sanger sequencing?

  • ddNTPs bind to the DNA chain and terminate extension
  • This creates DNA fragments of varying lengths
  • The fragments are then separated by size
• What method uses chain termination during DNA replication?

  The Sanger method
• What does the image show about the relationship between DNA fragment size and distance migrated?

  Larger DNA fragments migrate shorter distances than smaller fragments
• What is the first step in the DNA amplification process?

  Denaturation at 96°C separates DNA strands
• What is the purpose of the phosphodiester linkage shown in the image?

  It connects the nucleotides in the synthesized strand
• How does the dideoxynucleotide stop the synthesis of the strand?

  • The dideoxynucleotide lacks a 3' hydroxyl group
  • This prevents the formation of a new phosphodiester bond
  • So the strand synthesis is terminated
• How many times does the DNA amplification cycle typically repeat?

  • 25 to 35 times
  • Each cycle doubles DNA copies
  • Results in millions of identical fragments
• How does the DNA replication process work to determine the DNA sequence?

  • The DNA strand to be sequenced is primed
  • Four separate reactions are prepared, each with a different replication-stopping nucleotide (A, T, C, G)
  • The replication products from the 'C' reaction are separated by electrophoresis
  • The sequence is read as complementary bands containing the labeled strands
• What are the main reagents used in Sanger sequencing?

  • Template DNA
  • Polymerase
  • dNTPs
  • Primers
• How does the dideoxynucleotide stop the synthesis of the DNA strand?

  • The dideoxynucleotide lacks a 3' hydroxyl group
  • This prevents the formation of a new phosphodiester bond
  • So the strand synthesis is terminated
• What temperature is used for annealing in DNA amplification?

  55°C
• What is the name of the molecule shown on the left side of the image?

  Synthesized strand
• How does the structure of the template strand differ from the synthesized strand?

  The template strand has a continuous sugar-phosphate backbone, while the synthesized strand has phosphodiester linkages
• What enzyme is involved in the Sanger method of DNA sequencing?

  DNA polymerase
• What does the graph in part B) show about the relationship between DNA fragment size and distance migrated?

  The graph shows an inverse relationship, where larger DNA fragments migrate shorter distances than smaller fragments
• What is the relationship between distance traveled and fragment size in gel electrophoresis?

  • Inverse relationship
  • Smaller fragments travel further
  • Larger fragments travel shorter distances
• How does capillary gel electrophoresis separate the DNA fragments in Sanger sequencing?

  The fragments are separated by size as they migrate through the gel
• What is the purpose of the polymerase enzyme in Sanger sequencing?

  To extend the DNA chain during primer annealing