Sanger sequencing

Summary

Sanger sequencing is a method used to determine DNA sequences by incorporating modified nucleotides, called dye-deoxynucleotides, which halt DNA chain extension. The classical method, developed in the 1980s, used four separate reaction tubes, each with a different dye-deoxynucleotide. Modern Sanger sequencing utilizes fluorescent dyes and a single reaction tube, with DNA fragments separated by capillary electrophoresis and detected via fluorescence. This method remains essential in DNA sequencing due to its accuracy and ability to sequence small DNA fragments.

Takeaways

• 😀 Sanger sequencing is a method for determining the sequence of DNA by using dideoxynucleotides (ddNTPs) to terminate the elongation of DNA strands.
• 😀 The basic principle of Sanger sequencing involves adding ddNTPs to a growing DNA chain, causing the polymerization process to stop when incorporated.
• 😀 Classical Sanger sequencing uses four separate reaction tubes, each with one type of ddNTP and a radioactive label to detect terminated fragments.
• 😀 Each tube in classical Sanger sequencing corresponds to one of the four nucleotides (A, T, C, G) and allows the identification of the DNA sequence.
• 😀 Modern Sanger sequencing uses fluorescently labeled ddNTPs instead of radioactive labels, enabling the reaction to be conducted in a single tube.
• 😀 Fluorescently labeled ddNTPs in modern sequencing allow different nucleotides to be detected by their unique color emissions.
• 😀 Capillary electrophoresis is used in modern Sanger sequencing to separate DNA fragments, followed by detection of fluorescence signals by CCD devices.
• 😀 The removal of the 3' hydroxyl group in ddNTPs is what prevents further elongation of the DNA strand during sequencing.
• 😀 Polyacrylamide gel electrophoresis in classical sequencing was used to separate fragments by size, while modern systems rely on capillary electrophoresis.
• 😀 Fluorescent-based detection systems in modern Sanger sequencing enable faster, more efficient, and automated reading of DNA sequences compared to the older radioactive method.

Q & A

What is Sanger sequencing and how does it work?

Sanger sequencing is a method used to determine the sequence of DNA. It works by incorporating modified nucleotides called dideoxynucleotides (ddNTPs), which lack a 3' hydroxyl group, causing the DNA polymerization process to stop when they are added to the growing DNA strand.

What are the three main parts of Sanger sequencing covered in the video?

The three main parts of Sanger sequencing discussed are: 1) the principle of the method, 2) classical Sanger sequencing used in the 1980s, and 3) modern Sanger sequencing using fluorescent dyes.

What is the key difference between dNTPs and ddNTPs in Sanger sequencing?

dNTPs (deoxynucleotide triphosphates) are the normal building blocks for DNA synthesis, whereas ddNTPs (dideoxynucleotide triphosphates) lack a 3' hydroxyl group, preventing the addition of further nucleotides and thus causing the polymerization reaction to stop.

Why does the reaction stop when ddNTPs are incorporated into the DNA sequence?

The reaction stops because ddNTPs lack a 3' hydroxyl group, which is necessary for the formation of the phosphodiester bond between nucleotides. Without this hydroxyl group, DNA polymerase cannot add the next nucleotide, terminating the chain elongation.

What is the purpose of using four separate tubes in classical Sanger sequencing?

In classical Sanger sequencing, four separate tubes are used, each containing one specific ddNTP (ddATP, ddTTP, ddGTP, ddCTP) at a low concentration. This allows each tube to randomly terminate the DNA sequence at different points, depending on which ddNTP is incorporated.

How does polyacrylamide gel electrophoresis help in classical Sanger sequencing?

Polyacrylamide gel electrophoresis is used to separate the DNA fragments generated by chain termination at different lengths. The resulting gel allows the visualization of the fragments, helping to determine the DNA sequence based on fragment size.

What is the advantage of using fluorescent dyes in modern Sanger sequencing?

Fluorescent dyes allow all four reactions (ddATP, ddTTP, ddGTP, ddCTP) to occur in a single tube, unlike in classical sequencing where four separate tubes were required. Each ddNTP is labeled with a different color, making it easier to identify and analyze the sequence in one reaction.

What role does capillary electrophoresis play in modern Sanger sequencing?

Capillary electrophoresis is used to separate the fluorescently labeled DNA fragments in a narrow capillary tube. This method is more efficient than traditional gel electrophoresis and allows for faster and higher-resolution analysis of the sequencing results.

What technology is used to detect the fluorescent signals in modern Sanger sequencing?

The fluorescent signals are detected using a CCD (charge-coupled device), which captures the light emitted by the fluorescent dyes as the fragments pass through the capillary during electrophoresis.

How has the Sanger sequencing method evolved over time?

Sanger sequencing has evolved from using radioactive labels and four separate reaction tubes to using fluorescent dyes and capillary electrophoresis. These advancements have made the process more efficient, automated, and capable of producing results faster and with greater accuracy.