Why are dideoxynucleoside triphosphates required for Sanger DNA sequencing?

Dideoxynucleoside triphosphates (ddNTPs) serve as the pivotal molecules in the chain termination method of Sanger DNA sequencing. Unlike regular nucleotides, ddNTPs lack a critical hydroxyl group on the 3' carbon of the sugar ring, having a hydrogen atom in its place. This slight modification is what allows them to halt the synthesis of DNA.

When DNA polymerase attempts to add a ddNTP to the growing DNA strand, it's unable to form the necessary phosphodiester bond for chain elongation due to the missing hydroxyl group. Consequently, the DNA strand ends at the point of ddNTP incorporation. By carefully regulating the proportion of ddNTPs to regular deoxynucleoside triphosphates (dNTPs), the sequencing reaction produces DNA fragments of varying lengths, each terminated at different nucleotide positions by ddNTPs. These fragments form the basis for determining the DNA sequence.

DNA polymerase is the enzyme responsible for the replication of DNA. Its primary role within the context of Sanger DNA sequencing is to synthesize a new strand of DNA by adding nucleotides to the 3' end of a primer, which is complementary to the template strand. It does this by reading the template and incorporating the corresponding nucleotides.

In Sanger sequencing, DNA polymerase must accurately choose between adding a regular dNTP or a ddNTP. The success of this sequencing technique hinges on the enzyme's faithful yet selective addition of nucleotides, as this determines the synthesis of DNA fragments that will be terminated at specific locations due to the inclusion of ddNTPs. It's also worth mentioning that the choice of DNA polymerase is crucial for the accuracy and efficiency of the sequencing since different polymerases exhibit different processivity and fidelity characteristics.

Chain termination is a critical event in Sanger DNA sequencing that results from the incorporation of a ddNTP into the growing DNA strand. This process effectively 'caps' the strand, preventing further elongation. The genius of Sanger's method lies in leveraging this termination mechanism to create a series of DNA fragments that terminate at every possible nucleotide along the template strand.

Understanding Chain Termination

By including a small, controlled quantity of ddNTPs in the reaction, DNA polymerase occasionally incorporates these modified nucleotides rather than the standard ones. The DNA strands synthesized are essentially snapshots that end at different points, reflecting the base sequence of the template. By analyzing the lengths and terminal nucleotides of these fragments, we can construct the sequence of the original DNA.

Gel electrophoresis is the downstream technique used in Sanger DNA sequencing to analyze the DNA fragments produced by chain termination. During electrophoresis, an electric field is applied across a gel matrix, compelling the negatively charged DNA fragments to migrate towards the positive electrode.

Separation by Size

Smaller fragments move faster than larger ones, resulting in a separation by size. This process ultimately creates a series of discrete bands, each representing DNA strands that have terminated at different lengths. Because the DNA fragments differ by only a single nucleotide, the bands on the gel form a ladder-like pattern. The sequence of DNA can be read by interpreting this pattern from the smallest to the largest fragment. Gel electrophoresis is a powerful tool not only for DNA sequencing but also for the general analysis and purification of nucleic acids.