Do Special Replication and Tagging


The first step in DNA sequencing is similar to replicating (making copies of) DNA.

  • Start off with a strand of DNA to copy. This is called a template. To this, you add a mix of free nucleotides- As, Ts, Cs, and Gs, and you a short strand (about 20-30 nucleotides) of DNA called a primer. This will attach to the template and allow you to add free nucleotides. You also need an enzyme to do all the work.

  • You begin the reaction by heating. The two strands of DNA will separate, then the primer will stick to its intended location. The enzyme, called DNA polymerase, will elongate the primer by adding free nucleotides one-by-one. If you allowed the reaction to go to completion at this point, you would end up with a new strand of DNA that is a copy of your template.

  • This sounds very much like replication, but in sequencing there is one difference. When you sequence, some of the free nucleotides aren't deoxynucleotides, but dideoxynucleotides. Dideoxynucleotides are just like regular nucleotides, except that they have no 3' hydroxyl group. This means that once they are added to the end of a DNA strand, there's no way to continue elongating it and the process of replication stops. In sequencing, just a few (about 5&) of all the free nucleotides in solution are dideoxynucleotides,

  • Let's say we used dideoxy thymine (ddT) in our replication. Most of the time when a 'T' is required to make the new strand, the enzyme will attach oridinary deoxynucleotide nucleotide and will go ahead and add more nucleotides. However, 5& of the time, the enzyme will get a dideoxy-T and stop elongating. The partial strand of DNA will eventually break away from the enzyme and template, a dead end product.

  • I Because the primer always attaches to template at the same place, all the strands made in the solution will start in the same position. However, all of the copies of DNA being made in the solution will get terminated by a dideoxy 'T". Because there are both deoxy- and dideoxy-Ts, the copied strands will be a variety of different lengths, stopping at every T in the sequence. To find out where all the T's are in the original strand, all we have to do is find out the sizes of all the terminated products!

  • Luckily we have a process to separate these strands by size------Gel Electrophoresis!

  • Geneticists tag the ddT with radioactive dye so that each length that ends with dideoxy-T it can then be seen on a gel plate.

  • To sequence, dyed ddA, ddG and ddC are also added, producing strands of different lengths that end in a tagged dideoxynucleotide. So, on a gel we can read the sequence based on the pattern of tagged fragments.

  • Go to Gel Electrophoresis to see what the above sequence would look like on a gel and to learn more about Gel Electrophoresis.