Dideoxy nucleotides are used in which of the following techniques? a) RT-PCR b) DNA Sequencing c) Gene cloning d) restriction mapping
The correct answer and explanation is:
The correct answer is b) DNA Sequencing.
Dideoxy nucleotides are primarily used in DNA sequencing, specifically in the Sanger sequencing method. This technique, developed by Frederick Sanger in the 1970s, revolutionized the study of genetics and genomics by providing a method for determining the precise order of nucleotides (A, T, C, and G) in a DNA molecule.
The key feature of the Sanger sequencing method is the incorporation of dideoxynucleotides (ddNTPs) along with regular deoxynucleotides (dNTPs) during DNA synthesis. Dideoxynucleotides are chain-terminating agents because they lack a hydroxyl group (-OH) at the 3′ carbon of the sugar ring. This prevents further elongation of the DNA strand once a dideoxynucleotide is incorporated into the growing DNA chain.
During the sequencing reaction, a mixture of normal dNTPs (which are required for strand elongation) and a small proportion of labeled ddNTPs is added. Each ddNTP is specific to one of the four bases—adenine (A), thymine (T), cytosine (C), or guanine (G). As the DNA polymerase extends the DNA strand, occasionally, a ddNTP is incorporated, causing termination of the chain at that position. By running the resulting fragments on a gel or using capillary electrophoresis, the length of each fragment reveals the position of that particular base in the DNA sequence.
This process allows the determination of the exact sequence of nucleotides in the DNA sample. Sanger sequencing is highly accurate and was once the standard method for DNA sequencing, though newer high-throughput sequencing technologies have largely replaced it for large-scale genome sequencing.
In contrast, techniques like RT-PCR, gene cloning, and restriction mapping do not rely on dideoxy nucleotides. RT-PCR amplifies RNA sequences, gene cloning is used to replicate genes in a host organism, and restriction mapping identifies the positions of restriction enzyme cut sites on a DNA molecule.