Complementary Strand Synthesis:

The second strand synthesis is started by adding enzyme to a reaction mixture containing single strand DNA template with 5' radiolabelled DNA primers that are complementary to single stranded template, a mixture of dNTP with its dideoxydNTP form and the remaining three dNTPs. At a time four parallel reaction performed with different dideoxynucleotide i.e. dideoxyATP, dideoxyTTP, dideoyGTP, dideoxyCTP for each reaction. The concentration of dideoxyNTP should be only 1% of that particular dNTP to get a whole series of labelled strands ends up with that dideoxy NTP. The lengths of these strands are dependent on the location of the base relative to the 5' end (Fig. 3).

Figure 3. Growing Daughter strand with reference to the template nucleotide.