How Sanger Sequencing Works? (Classic Sanger Method)

Summary

This video script delves into DNA sequencing, highlighting the foundational Sanger method developed in 1977. It explains the necessity of a DNA template, primer, nucleotides, and DNA polymerase for in vitro DNA synthesis. The Sanger technique utilizes dideoxynucleotides (ddNTPs) to terminate DNA synthesis, creating fragments that are separated by size using polyacrylamide gel electrophoresis. The sequence is determined by reading the autoradiograph from bottom to top, revealing the DNA's 5' to 3' sequence. The script provides a comprehensive understanding of DNA sequencing, essential for those interested in molecular biology.

Takeaways

• 🧬 DNA sequencing is a technique used to determine the precise order of nucleotides in a DNA segment.
• 🔬 The Sanger method, developed in 1977, is a foundational technique for modern DNA sequencing.
• 📚 In vitro DNA synthesis is central to the Sanger method, relying on the principles of DNA replication.
• 🌟 Key components for in vitro DNA synthesis include a DNA template, primer, nucleotides, and DNA polymerase.
• 🔑 The primer is essential as it provides the 3' hydroxyl group needed for DNA polymerase to start chain synthesis.
• 🔄 Dideoxy (dd) nucleotides are modified nucleotides that, when incorporated, terminate DNA synthesis due to the lack of a 3' hydroxyl group.
• 🧪 Sanger sequencing involves four separate reactions, each with a different ddNTP to cause chain termination at varying points.
• 📈 The resulting fragments are separated by size using polyacrylamide gel electrophoresis, with smaller fragments migrating further.
• 📸 Autoradiography is used to visualize the fragments, with the sequence determined by reading the bands from the smallest to the largest.
• 🔄 The sequence obtained from the autoradiograph represents the newly synthesized strand, which is complementary to the template strand.

Q & A

What is DNA sequencing?

DNA sequencing is a technique used to determine the precise order of nucleotides within a DNA segment.

Who developed the Sanger method of DNA sequencing?

The Sanger method of DNA sequencing was developed by Frederick Sanger and colleagues at the University of Cambridge in 1977.

What is the basis of the Sanger sequencing technique?

The Sanger sequencing technique is based on the principle and biochemistry of DNA replication and involves in vitro DNA synthesis.

What are the basic requirements for in vitro DNA synthesis?

The basic requirements for in vitro DNA synthesis include a DNA template strand, a primer, nucleotides, and DNA polymerase.

Why is a primer necessary for DNA synthesis?

A primer is necessary because DNA polymerase requires a three prime hydroxyl group to form a phosphodiester bond with the incoming deoxynucleotide, which is provided by the primer.

What are dideoxynucleotides (ddNTPs) and how do they function in DNA sequencing?

Dideoxynucleotides (ddNTPs) are modified deoxynucleotides that lack a three prime hydroxyl group, causing DNA synthesis to terminate when incorporated. They are used in Sanger sequencing to create a set of terminated DNA fragments.

How does the Sanger sequencing technique differ from other sequencing methods?

The Sanger sequencing technique, also known as the chain termination method, uses ddNTPs to terminate DNA synthesis at random points, creating a ladder of fragments that can be used to determine the DNA sequence.

What are the four separate reactions involved in Sanger sequencing?

The four separate reactions in Sanger sequencing involve the incorporation of one type of ddNTP (ddATP, ddCTP, ddGTP, or ddTTP) in each reaction, along with the standard dNTPs and other common components.

How are the results of Sanger sequencing analyzed?

The results of Sanger sequencing are analyzed by running the reaction products on a polyacrylamide gel electrophoresis, which separates the DNA fragments by size, and then reading the sequence from the autoradiograph of the gel.

What is the significance of the autoradiograph in Sanger sequencing?

The autoradiograph is a photograph of the gel produced by radiation from radioactive material present in the partial DNA fragments, which allows for the visualization and interpretation of the DNA sequence.

How is the DNA sequence determined from the autoradiograph?

The DNA sequence is determined by reading the bands on the autoradiograph from bottom to top, which corresponds to the sequence of the newly synthesized strand, and then determining the complementary sequence of the template strand.