Okay, let’s try to make this a bit more humorous. Here goes:
Sanger sequencing, also known as the dideoxy method, is a technique used in molecular biology to determine the nucleotide sequence of DNA. It’s named after Fred Sanger, who was awarded the Nobel Prize in Chemistry in 1980 for his work on the technique. That’s right, folks – this guy was a real “Sanger” of sequencing!
Sanger sequencing is based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) into growing DNA strands during replication. DNA polymerase, the enzyme that’s responsible for synthesizing new strands of DNA, normally incorporates nucleotides into a growing strand by adding them to the 3′ end. But when a ddNTP is present, it can be incorporated but it can’t be further extended, resulting in the termination of DNA synthesis. In other words, the ddNTP is like the “mom” of nucleotides – it says “No more DNA for you!” and shuts down the whole operation.
To perform Sanger sequencing, a small amount of DNA is first amplified using the polymerase chain reaction (PCR) to produce many copies of the target sequence. This amplified DNA is then fragmented into smaller pieces, kind of like breaking a really long novel into manageable chapters. Each fragment is then used as a template for a separate DNA synthesis reaction. In each reaction, a mixture of all four regular nucleotides (A, C, G, and T) is added, along with a small amount of a single ddNTP. This results in the synthesis of many DNA strands, each of which terminates at a specific point where the ddNTP was incorporated. It’s like a high school English class where each student has to write a different paragraph of a story, but they all have to stop at the same point.
These newly synthesized DNA strands are then separated by size using a technique called gel electrophoresis. This separates the strands based on their size, with smaller strands migrating faster through the gel. It’s kind of like a school field trip to a water park, where the kids who are smaller and lighter can slide down the water slides faster than the bigger kids. The DNA strands are then transferred to a nylon or nitrocellulose membrane, where they are exposed to a radioactively labeled or fluorescently labeled nucleotide. This labeled nucleotide will bind only to the complementary nucleotide on the DNA strands, allowing the sequence to be visualized using X-ray film or a specialized imaging system. It’s like a fancy dating app that only matches you with people who have complementary DNA sequences.
Sanger sequencing is still widely used today, although it has been largely replaced by more efficient next-generation sequencing techniques. It remains a powerful tool for sequencing smaller, specific regions of DNA and is still used in many research and clinical laboratories. So even though it’s not the newest kid on the block, Sanger sequencing still has plenty of “DNA” in the game!