Question

Briefly, how does the chain termination method work? Make sure to discuss the role of ddNTPs.

Answer 1

Sanger sequencing are called as chain termination method which can sequence regions of DNA up to about 900 base pairs in length.

At first the DNA sample to be sequenced will be mixed along with the primer, DNA polymerase, DNA nucleotides (dATP, dTTP, dGTP, and dCTP) and the respective dye-labeled dideoxy nucleotide.

Then the template DNA is denatured to separate the strands, so that the primer can bind to the single-stranded template. Once bounded, the DNA polymerase starts synthesize new DNA starting from the primer. DNA polymerase adds nucleotides to the chain until the dideoxy nucleotide(ddNATPs) is added instead of regular nucleotide. After the ddNATPs is added to the chain no further nucleotides can be added to it as there is no hydroxyl present in the dideoxy nucleotide which acts as hook for the further nucleotide to connect.

After repeating this process a number of times, the dideoxy nucleotide would have been incorporated to every single position of the target DNA. The fragments from those will be labelled at the ends with dyes that indicate their final nucleotide.

Then the fragments will run through a process called capillary gel electrophoresis where the dyes will be illuminated by a laser and will be detected by a detector and the peaks are recorded through chromatogram.The DNA sequence is read from the peaks in the chromatogram.

Dideoxy nucleotides(ddNTPs) lacks a hydroxyl group on the 3’ carbon of the sugar ring which acts as a hook allowing a new nucleotide to add on an existing chain.

Hence when a dideoxy nucleotide was added to the chain, which has no hydroxyl no further nucleotides can be added and the chain ends.