SOLiD DNA sequencing is a very complicated process, so just remember it uses an enzyme called ligase. Ligase, unlike DNA polymerase, does not add individual bases. Instead, it adds a whole segment of bases that is complementary to the DNA being sequenced. Also, remember that it has a built-in proofreading capability which gives it very low error rates. We'll gloss over some of the finer details.

This diagram illustrates steps 3 and 4, in which ligase adds a short complementary DNA fragment in the left column, and then shifts one base over in the right column.


This diagram shows the assignment of the four colors to each two-base combination.

  1. Emulsion PCR is performed on all the DNA fragments to be sequenced.
  2. Each DNA fragment base pairs to a short starting sequence which starts off the sequencing. Along with the DNA fragment and short starting sequence called a primer, all possible eight-base DNA fragments are added. This means 4^8, or over 65,000 different DNA fragments! Each modified eight-base DNA fragment also has a fluorescing group whose color can be detected.
  3. Ligase adds the correct eight-base DNA fragment complementary to the DNA being sequenced, and the color of the fluorescing group is recorded before the last three bases, along with the dye, is cleaved off.
  4. This process is repeated until the DNA has been completely sequenced. At this point, you may have realized that one sequencing cycle only sequences one out of every 5 bases. This problem is solved by removing the new DNA copy along with one base from the original primer, shifting the reading frame one base back. This process is repeated four times.

Now, we have data of four different colors. To make things more complicated, although you would logically think each color should correspond to one base, each color actually corresponds to TWO bases, the two bases at the beginning of each DNA fragment, even though there are only FOUR different colors. If you do the math, you will find that there really should be SIXTEEN different colors to correspond to every possible two-base combination (4 times 4, since there are 4 possible bases per position). However, this problem is solved by assigning four different two-base combinations to each of the four different colors. Since we had five different reading frames, by doing some complex analysis, we are able to determine the sequence from this data, while at the same time double-checking the sequencing several times. (47)

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