define dideoxysequencing
Dideoxysequencing, also known as Sanger sequencing, is a method for determining the order of DNA sequences. It involves the use of modified nucleotides called dideoxynucleotides (ddNTPs) that lack a hydroxyl group on the 3' carbon of the sugar molecule. These ddNTPs are incorporated into a growing DNA strand during polymerization, but cause chain termination as they lack the necessary hydroxyl group for the next nucleotide to attach. By using fluorescently-labeled or radiolabeled ddNTPs, the terminated fragments can be separated by size through gel electrophoresis and the sequence can be determined by analyzing the order of the fragments produced. This method played a key role in the Human Genome Project and continues to be used for DNA sequencing in research and medical applications.
Dideoxysequencing, also known as Sanger sequencing, is a widely used method for DNA sequencing. It was developed by Frederick Sanger and his colleagues in the late 1970s. This technique allows for the determination of the order of nucleotides (adenine, cytosine, guanine, and thymine) in a DNA molecule.
In dideoxysequencing, a DNA template is mixed with a primer, DNA polymerase enzyme, and four types of nucleotides (A, C, G, and T). Additionally, small amounts of chain-terminating dideoxynucleotides (ddNTPs) are included. These ddNTPs lack a 3'-OH group, which is necessary for the formation of phosphodiester bonds that link the nucleotides together.
As the DNA polymerase enzyme extends the primer, it occasionally incorporates a ddNTP instead of a regular nucleotide. This ddNTP acts as a chain terminator because it lacks the 3'-OH group, preventing further extension of the DNA strand. By including all four ddNTPs in separate reactions, each terminating at a specific nucleotide (A, C, G, or T), a set of DNA fragments of different lengths is generated.
The mixture of DNA fragments is then separated based on their size using gel electrophoresis. The fragments are loaded onto a gel matrix and subjected to an electric field, causing them to migrate through the gel. Smaller fragments move faster through the gel, while larger fragments move slower. The migration distance of each fragment is detected using fluorescent or radioactive labels.
After the gel electrophoresis, the DNA fragments are visualized and read in order to determine the DNA sequence. The sequence is read by analyzing the relative positions of the different length fragments corresponding to each nucleotide. By analyzing the separate reactions for each nucleotide, the sequence of the original DNA molecule can be determined.
Overall, dideoxysequencing is a widely used and highly accurate method for DNA sequencing, providing valuable information for genetic research, DNA fingerprinting, and various diagnostic applications.