ddNTPs and DNA Sequencing

Hydroxyl Group and Nucleotide Addition

• The hydroxyl group ($\text{OH}$) is crucial for nucleotide addition.
• New nucleotides are added to the $\text{OH}$ group, which allows for strand extension.
• If the $\text{OH}$ group is blocked, no further nucleotides can be added to that strand.

Synthetic ddNTPs

• Synthetic dideoxynucleoside triphosphates (ddNTPs) are used with DNA samples.
• These ddNTPs attach to the DNA strand where replication is occurring.
• This mimics natural replication, where a complementary sequence is created by adding new deoxynucleoside triphosphates (dNTPs).
• However, the process is done artificially.
• A single strand is synthesized at the 3' end.
• Bases are added sequentially as the synthesis progresses.

Chain Termination with ddNTPs

• The reaction requires: DNA polymerase, ligase, and appropriate temperature.
• When a ddNTP, which is a dye-deoxyribonucleotide, is incorporated into the growing strand, extension stops.
• Each ddNTP is labeled with a specific dye (A, T, C, or G).
• The process iterates through samples, and each time a ddNTP is incorporated, the extension halts.
• The sequence is read based on the order of ddNTP incorporation and termination.