2',3'-Dideoxynucleotide Triphosphate (ddNTP)
Definition
A nucleotide analogue lacking the 3'-hydroxyl group present in normal dNTPs. When incorporated by DNA polymerase into a growing chain, it prevents further extension because no 3'-OH is available for the next phosphodiester bond. The selectivity of ddNTP incorporation, at every occurrence of a given base, is the mechanism by which Sanger sequencing reads a sequence.
- Structural difference
- Lacks the 3'-hydroxyl group present in dNTPs
- Effect on synthesis
- Terminates the DNA chain once incorporated
- Used in
- Sanger sequencing chemistry
Common questions
Why does the missing 3'-OH group stop DNA synthesis at that point?+
DNA polymerase needs a free 3'-hydroxyl on the last incorporated nucleotide to form the phosphodiester bond with the next one; without it, no further nucleotides can be added and the growing strand ends there.
How does incorporating ddNTPs into a reaction actually generate readable sequence data?+
A mixture of normal dNTPs with a small proportion of fluorescently labelled ddNTPs produces a population of fragments that terminate at every occurrence of a given base; separating these fragments by size with capillary electrophoresis and reading the label at each length reveals the base order.
Related terms
- Big Dye Terminator (BDT) V3.1
- Applied Biosystems' (now Thermo Fisher Scientific's) commercial cycle sequencing kit using four spectrally distinct dichlororhodamine dyes coupled to the four ddNTPs. The...
- Cycle Sequencing
- A Sanger sequencing format using a thermostable DNA polymerase and repeated denaturation-annealing-extension cycles (typically 25-35 cycles). Produces exponentially more termination products from...
- EMPOP (ENBISI Mitochondrial Population Database)
- The ENFSI-curated reference database of forensic-grade mtDNA haplotypes used to estimate the frequency of a given control-region profile in a specified population....
- Phred Quality Score
- A logarithmic estimate of base-calling error probability (Phred Q = -10 log10 P). Q20 = 1% error rate; Q30 = 0.1% error...
- rCRS (Revised Cambridge Reference Sequence)
- The standard reference sequence for the human mitochondrial genome (NC_012920.1, 16,569 bp), used as the coordinate system for reporting all forensic mtDNA...
- Sequencing-by-Synthesis (SBS)
- The Illumina detection paradigm: reversibly fluorescent 3'-blocked nucleotides are incorporated one cycle at a time into clonally amplified clusters on a flow...