cloning vs. direct sequencing - (Mar/19/2007 )
can someone give me some advatages and disadvantages of cloning vs. direct sequencing of a PCR product.
direct sequencing gives you the average sequence of the population of PCR products.
thus the following situation is possible:
every single molecule in the population to has a single base error. However as the mistakes occur at random, the probability of the same mistake occuring at one base pair is low.
Thus the average sequencing result would show that the correct sequence even though every single molecule has a mistake. And since only a total correct molecule is any good to your expression work, the sequencing result would be missleading.
sequencing a cloned PCR product avoids the above problem. The tradeback would be more time expended before sequencing.
However considering the severity of the above problem, one should always sequence plasmid that been liagted to PCR inserts. Ie do your ligation work then seqeuence the PCR product within your plasmid.