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