Current technology allows sequencing of DNA fragments up to about 1,000 bps. There are two main strategies to obtain the fragments which go into sequencing machine. The original one and still widely used is the shotgun method where large DNA fragments are randomly broken into smaller fragments, which in turn are amplified and sequenced. The size of the smaller fragments is usually about 1,500 kb, so each of them ends up being only partially sequenced. Since the procedure is random, one needs to ensure that the original fragment was entirely sequenced by creating a very large number of small fragments.
The directed strategy is harder to implement but requires considerably much fewer sequencing reactions. Currently, a transposon--based directed sequencing strategy is implemented at LBNL and Stanford. The following is a brief description of how it works.
First, the large fragments are broken into small fragments of about 3,300 kb long on average (mechanical shearing is used at LBL to break the bonds). Then the ends of each of the small fragments (we will refer to them as subclones or 3kbs) are sequenced ( end-sequencing process), and the optimal path though the entire original fragment is found by looking at how the 3kbs overlap. We want the path such that when each of the chosen 3kbs is completely sequenced, we will end up with the entire original clone, sequencing the smallest possible number of subclones. The procedure is called path--building.
Once the set of subclones to sequence is chosen (if the original
fragment has the length of about 80kb, we usually end up with about 25
subclones), we use the technique developed at LBNL. The technique is
transposon--based and allows to randomly insert ``jumpable''
elements called transposons into a fragment. Then the approximate location of each of
the transposons is found using PCR (see week 9 lecture
on PCR) and the optimal path which spans the entire 3kb is found. (The
chosen transposons are spaced about 300bps away from each other.) Each
of the transposons
contain a primer (see later) and it allows us to sequence the fragment
in both directions, starting from a point where a chosen transposon is
inserted. If everything goes well we will end up with one entirely
sequenced 3kb, completely double--stranded i.e. covered with reads
going in both directions at any position along the subclone.