Sanger Sequencing Principles

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