what is dideoxysequencing method
Dideoxysequencing, also known as Sanger sequencing, is a method used to determine the sequence of nucleotides in a DNA molecule. The method involves using a DNA polymerase enzyme, DNA primers, fluorescently labeled dideoxynucleotides, and a template DNA strand.
The DNA polymerase enzyme extends a DNA primer by adding complementary nucleotides to the template strand, and when a dideoxynucleotide is incorporated into the growing DNA strand, DNA synthesis stops. This results in the creation of fragments of different lengths, each terminating with a dideoxynucleotide. The fragments are then separated by size using gel electrophoresis, and the sequence is determined by analyzing the pattern of fluorescent signals generated by the different colored dideoxynucleotides incorporated into the fragments.
This method was developed by Frederick Sanger in the 1970s and is widely used in molecular biology research and genetic analysis.
The dideoxysequencing method, also known as Sanger sequencing, is a technique used to determine the order of nucleotides (A, T, C, G) in a DNA sequence. It was developed by Frederick Sanger and his colleagues in the late 1970s.
Here is a step-by-step explanation of the dideoxysequencing method:
1. DNA Template Preparation: The DNA fragment that needs to be sequenced is isolated, purified, and amplified using the polymerase chain reaction (PCR) technique. This generates a large population of identical DNA fragments.
2. Primer Annealing: A short synthetic DNA primer, complementary to a known region of the DNA template, is added to the reaction mixture. The primer binds to the DNA template at its specific binding site.
3. DNA Polymerase Reaction: A DNA polymerase enzyme is added, along with the four deoxyribonucleotide triphosphates (dNTPs) that serve as the building blocks for DNA synthesis. One of these dNTPs is labeled with a fluorescent dye, allowing the final DNA products to be visualized.
4. Dideoxynucleotide Incorporation: Along with the four regular dNTPs, small amounts of dideoxynucleotide triphosphates (ddNTPs) are added to the reaction mixture. Unlike dNTPs, ddNTPs lack the hydroxyl group (-OH) at the 3' carbon of the sugar molecule, preventing further DNA synthesis when incorporated into the growing DNA strand.
5. DNA Fragment Separation: The reaction mixture is divided into four separate tubes, each containing one of the four ddNTPs (A, T, C, G). These tubes undergo gel electrophoresis, which separates the DNA fragments based on their size. The gel is composed of a matrix that s the progress of the DNA fragments as they migrate through it.
6. Visualization and Analysis: After electrophoresis, the DNA fragments are visualized using a technique such as autoradiography or fluorescence detection. A DNA sequencer reads the fluorescence signals emitted by the labeled fragments as they pass through a detector. The sequence is then determined based on the order of the different color peaks obtained.
By repeating these steps multiple times, using different DNA templates or primers, the entire DNA sequence can be determined. This method revolutionized DNA sequencing and played a crucial role in many significant scientific discoveries and advancements in the field of genetics.