6.3.1 DNA sequencing

• What is the purpose of DNA sequencing?

  Determining nucleotide order
• Sanger sequencing is highly accurate for short DNA sequences.


  True
• The Sanger method relies on dideoxynucleotides (ddNTPs) to terminate DNA elongation at specific nucleotides
• Steps in Sanger sequencing

  1️⃣ Prepare the template DNA and primer

  2️⃣ Mix template DNA, primer, dNTPs, ddNTPs, and DNA polymerase

  3️⃣ Incubate at various temperatures

  4️⃣ Separate fragments by gel electrophoresis

  5️⃣ Determine sequencing by band order
• ddNTPs ensure full coverage of the template DNA in Sanger sequencing.

  True
• Match the sequencing method with its key feature:

  Sanger sequencing ↔️ Chain termination

  Next-generation sequencing ↔️ Parallel sequencing
• What is the primary sequencing method used in Illumina sequencing?

  Sequencing by synthesis
• What are the normal nucleotides used for DNA synthesis?

  dATP, dCTP, dGTP, dTTP
• Steps in Sanger sequencing

  1️⃣ Prepare the template DNA and primer

  2️⃣ Mix the template DNA, primer, dNTPs, ddNTPs, and DNA polymerase

  3️⃣ Incubate at various temperatures to allow DNA extension

  4️⃣ Separate fragments by gel electrophoresis

  5️⃣ Determine the sequence from the band order
• What is the key principle of Illumina sequencing?

  Sequencing by synthesis
• Cluster generation in Illumina sequencing involves amplifying DNA fragments on a flow cell.


  True
• Compared to Sanger sequencing, Illumina sequencing has higher throughput but produces shorter read lengths.
• What is a major advantage of Sanger sequencing over Illumina sequencing?

  High accuracy for short sequences
• Sanger sequencing is highly accurate for sequencing short DNA fragments.


  True
• Steps in the Sanger sequencing process

  1️⃣ Mix template DNA, primer, dNTPs, ddNTPs, and DNA polymerase

  2️⃣ Incubate to allow DNA extension

  3️⃣ Separate fragments by gel electrophoresis

  4️⃣ Determine the sequence from the band order
• What is the underlying principle of Illumina sequencing?

  Sequencing by synthesis
• Illumina sequencing uses ddNTPs to determine the sequence of DNA fragments.

  False
• Steps in Illumina sequencing

  1️⃣ Library preparation

  2️⃣ Cluster generation

  3️⃣ Sequencing by synthesis
• Sanger sequencing has a lower throughput
• What is the typical read length of Sanger sequencing?

  500-1000 bases
• Match the sequencing method with its key feature:

  Sanger Sequencing ↔️ High accuracy for short sequences

  Illumina Sequencing ↔️ High throughput, shorter reads
• Genome mapping is crucial for personalized medicine.


  True
• Match the sequencing method with its application:

  Sanger Sequencing ↔️ Short sequence accuracy

  Illumina Sequencing ↔️ Genome sequencing
• Sanger sequencing uses **chain termination
• What is a key advantage of next-generation sequencing (NGS)?

  High throughput
• ddNTPs are modified nucleotides that terminate DNA synthesis.


  True
• Why are ddNTPs essential in the Sanger method?

  Terminate DNA synthesis
• Sanger sequencing is expensive for sequencing large genomes
• Steps in Sanger sequencing

  1️⃣ Prepare the template DNA and primer

  2️⃣ Mix template DNA, primer, dNTPs, ddNTPs, and DNA polymerase

  3️⃣ Incubate at various temperatures

  4️⃣ Separate fragments by gel electrophoresis

  5️⃣ Determine sequencing by band order
• Illumina sequencing requires library preparation to add adapters to DNA fragments.


  True
• ddNTPs are modified nucleotides used to terminate DNA synthesis in Sanger sequencing.
• The presence of ddNTPs in Sanger sequencing ensures random termination of DNA synthesis.


  True
• In Illumina sequencing, DNA is fragmented and adapters are added during library preparation.
• How is the sequence determined in Illumina sequencing?

  Detecting fluorescently-labeled nucleotides
• Match the sequencing method with its key feature:

  Sanger Sequencing ↔️ Chain termination with ddNTPs

  Illumina Sequencing ↔️ Sequencing by synthesis
• DNA sequencing is the process of determining the precise order of nucleotides in a DNA molecule.
• What are ddNTPs used for in the Sanger method?

  Chain termination
• Match the sequencing method with its limitation:

  Sanger Sequencing ↔️ Low throughput

  Illumina Sequencing ↔️ Shorter read lengths
• What type of nucleotides are used in Sanger sequencing?

  ddNTPs
• Illumina sequencing utilizes the method called sequencing