What is Sanger sequencing?

Sanger sequencing, also known as the dideoxy chain termination method, is a pioneering technique for determining the order of nucleotides (A, C, G, T) in a DNA molecule. Developed by Frederick Sanger and his colleagues in 1977, it paved the way for modern DNA sequencing methods.

What are the key features of Sanger sequencing?

• Chain Termination: Sanger sequencing utilizes specially modified DNA nucleotides with dideoxynucleotides (ddNTPs) lacking a 3' hydroxyl group. When a ddNTP is incorporated into the growing DNA chain, it terminates further elongation.
• Four Reactions: The method involves running four separate reactions, each containing one type of ddNTP along with the normal dNTPs and a DNA primer. Each reaction stops at a specific point based on the incorporated ddNTP, revealing the base at that position.
• Gel Electrophoresis: The fragments generated in each reaction are then separated by size using gel electrophoresis. By analyzing the banding pattern, the sequence of the DNA molecule can be determined.

What is the importance of Sanger sequencing?

• Foundation for Modern Sequencing: Sanger sequencing laid the groundwork for the development of faster and more efficient next-generation sequencing (NGS) technologies.
• Reliable and Accurate: It remains a highly reliable and accurate method for sequencing relatively short DNA fragments (up to ~900 base pairs).
• Validation Tool: Sanger sequencing is often used to validate results obtained from NGS due to its high accuracy.

What are applications of Sanger sequencing in practice?

• Verification of Plasmids and PCR Products: Sanger sequencing is used to confirm the identity and sequence of plasmids used in genetic engineering or PCR products for further analysis.
• Mutation Detection: It can be used to identify specific mutations in genes associated with genetic diseases.
• DNA Forensics: Sanger sequencing plays a role in forensic analysis by analyzing short tandem repeats (STRs) in DNA samples for identification purposes.

Practical example

A researcher wants to verify the sequence of a gene they have amplified using PCR. They can perform Sanger sequencing on the PCR product. By analyzing the banding pattern on the gel, they can confirm if the amplified fragment matches the expected sequence and identify any potential errors introduced during PCR.

Critical remarks

• Limited Throughput: Sanger sequencing is slow and laborious compared to NGS, making it less suitable for large-scale sequencing projects.
• Read Length Limitations: The readable sequence length is limited to a few hundred base pairs, restricting its use for whole-genome sequencing.
• High Cost: While less expensive than NGS per base sequenced, Sanger sequencing can be costly for larger projects due to its lower throughput.