- In Sanger sequencing, a mixture of normal deoxyribonucleotides (dNTPs) and modified dideoxyribonucleotides (ddNTPs) is used to control DNA chain elongation.
- Under normal circumstances, DNA polymerase adds dNTPs to the growing DNA strand. Each dNTP contains a hydroxyl group (-OH) at the 3′ carbon of its sugar, which allows the formation of a phosphodiester bond with the next incoming nucleotide. This 3′-OH group is essential for continuous chain elongation, and so when only dNTPs are present, the polymerase synthesizes a complete complementary strand without interruption.
- The role of ddNTPs becomes critical when they are added to the reaction in small amounts. Unlike dNTPs, ddNTPs lack a 3′-OH group. When DNA polymerase incorporates a ddNTP into the growing strand, the absence of the 3′-OH prevents the addition of any further nucleotides. This event is known as chain termination. Because ddNTPs are present in only a limited proportion compared to dNTPs, their incorporation happens randomly along the newly synthesized DNA strands.
- As a result, the sequencing reaction produces a collection of DNA fragments of varying lengths, each ending at a point where a ddNTP was incorporated. For instance, one fragment may end after the addition of a ddATP, another after a ddCTP, and so on. When these fragments are separated by size through capillary electrophoresis, the sequence of the template DNA can be determined based on the positions at which elongation stopped. Modern sequencing techniques take advantage of this by labeling each type of ddNTP with a different fluorescent dye, allowing the terminated fragments to be detected and read automatically.
- An important distinction between Sanger sequencing and PCR is the use of primers. While PCR requires two primers—one for each strand of the target sequence—Sanger sequencing uses only a single primer. This primer anneals to the template DNA and directs DNA polymerase to synthesize just one complementary strand. The incorporation of ddNTPs then generates a nested set of terminated fragments from this single starting point, which ensures that sequencing data reflects the order of nucleotides in the chosen strand of the DNA template.
- The mixture of dNTPs and ddNTPs, combined with the single-primer design, is crucial to the success of Sanger sequencing. If only dNTPs were used, elongation would never terminate, and no sequence information could be derived. On the other hand, if only ddNTPs were present, DNA synthesis would terminate almost immediately after the first nucleotide incorporation, producing fragments that are too short to reveal the full sequence. The balance between the two ensures random but complete coverage of every nucleotide position in the target DNA, which is the principle that makes Sanger sequencing effective and accurate.
