Step 3: The polymerease reaction is divided into 4 reactions.

Step 4: The polymerase reaction continues with 4 nucleotide and one ddNTPs. Synthesis is terminated at the specific ddNTPs (either A, G. C. T) to give DNA fragment of different length.

Figure 27.2: Different Steps in Di-deoxy Method

The polymerization reaction is analyzed on a high resolution polyacrylamide gel. The use of sequenase allow to perform sequencing of long DNA stretches where as original sanger method is more appropriate for short length DNA.