Posted 14 September 2010 - 03:39 AM
I disagree with Rsm (sorry)...
Sequencing a PCR reaction directly and sequencing a fragment cloned from a PCR reaction gives you two different pieces of data, which may or may not be the same.
When you sequence a PCR reaction directly, you get back the sequence of the majority of the population of amplicons, whereas when you sequence an individual cloned PCR amplicon, you get back the sequence of that particular amplicon. Due to the possibility of base incorporation errors during amplification, the sequence of the cloned amplicon might differ from that of the population.
As an example, let's say you have 1,000 amplicons of a 1,000 basepair fragment. Let us further say that each one of these amplicons has one base incorporation error, and that they're all in a different location. If you sequence such a reaction directly, you'll get back the true sequence of the fragment, because the single error in each position is overwhelmed by the correct base that exists in the other 999 amplicons. From this population, however, it would be impossible to clone a fragment that has the correct sequence.
It's like saying in a classroom, most students have brown eyes. But if you pick an individual student, he or she might have blue eyes -- you won't know until you look at the student you've picked what color eyes he or she has...
If your goal is to determine the sequence of a fragment as it exists on the chromosome, then sequencing the PCR reaction directly is okay. If your goal is to clone a fragment from this population and use it in further studies, you need to sequence the individual clone to insure it has no base incorporation errors.