In a dideoxy DNA sequencing experiment, four separate reactions are carried out to provide the replicated material for DNA sequencing gels. Reaction products are usually run in gel lanes labeled A, T, C, and G.

Why is incorporation of a dideoxynucleotide during DNA sequencing identified as a 'replication-terminating' event?

Key Concepts

Dideoxynucleotides

Dideoxynucleotides (ddNTPs) are modified nucleotides used in DNA sequencing that lack a hydroxyl group at the 3' carbon of the sugar. This structural modification prevents the addition of further nucleotides during DNA synthesis, effectively terminating the DNA strand elongation when incorporated. Their use is crucial in generating fragments of varying lengths that can be analyzed to determine the DNA sequence.

DNA Replication

DNA replication is the biological process by which a cell duplicates its DNA, ensuring that each daughter cell receives an identical copy of the genetic material. During replication, DNA polymerase adds nucleotides to a growing DNA strand. The incorporation of a dideoxynucleotide interrupts this process, leading to the termination of the strand, which is essential for sequencing as it allows for the determination of the sequence based on fragment lengths.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. In the context of DNA sequencing, the terminated fragments generated by dideoxynucleotide incorporation are loaded into a gel matrix and subjected to an electric field. Smaller fragments migrate faster than larger ones, allowing for the visualization of the different lengths of DNA strands, which correspond to the sequence of nucleotides in the original DNA template.