Doing Things One Step at a Time

The key to NGS sequencing-by-synthesis is to make the DNA polymerase work one step at a time. We start with the ssDNA and then add one nucleotide, like this:

Note that this example would have a primer, but we will leave it out for simplicity.

Step 1

5′-ACCGTACGTTGC-3′

……….…...….….3′-G-5′

We take a picture. The fact that we see a blue color tells us that the letter G was added at the end. We cannot see the G directly, only the blue color.

Step 2

Then we use chemical reactions to turn off the blue color:

5′-ACCGTACGTTGC-3′

……….…...….….3′-G-5′

Step 3

We then add the next nucleotide—or, more accurately, the DNA polymerase adds the next nucleotide that is complementary to the letter in the top string:

5′-ACCGTACGTTGC-3′

……….........…3′-CG-5′

This time, when we take a picture, we see only the green color, which tells us that the letter C was added.

Step 4

Again, we turn off the light:

5′-ACCGTACGTTGC-3′

……….........…3′-CG-5′

Step 5

The DNA polymerase then adds the next complementary nucleotide:

5′-ACCGTACGTTGC-3′

………..….....3′-ACG-5′

This time, the picture shows an orange color.

Continue the Process

We continue the above process - adding a colored nucleotide, taking a picture, and then turning off the color - until the end. Special devices carry out these chemical reactions automatically.