why do cloning before sequencing - (May/08/2006 )
hello,
i am new in this field and never done microbiology.... and now i am searching protocol for sequencing microorganism.... but i wonder why it need to CLONE the PCR product before sequencing but not do sequencing immediately after PCR just like doing that in human sample?
Thank you very much!
in my lab it was mainly a cost issue, with sequencing costing more money than simply cloning and selecting recombinant, which you only sequence when you are sure you have the clone.
also sequencing after you have the clone means you know what sequence is in you plasmid and what is going to be expressed and that no errors have occured or sequence lost during possible restriction digestion (multiple RE sites)
i am new in this field and never done microbiology.... and now i am searching protocol for sequencing microorganism.... but i wonder why it need to CLONE the PCR product before sequencing but not do sequencing immediately after PCR just like doing that in human sample?
Thank you very much!
Well, I don't think it has got to do with the kind of tissue sample.
I was told that the sequencing quality is better when you use plasmids as matrices for the sequencing instead of raw pcr-products. Maybe the DNA-polymerase isn't so easy to fall off from a plasmid?!?
I think the long and short of it is that if you sequence from a PCR product, you will lose the first ~50-75 bp. I am can't remember why, but I think it has to do with primer artifacts and unincorporated dye terminators shielding small products. If you sequence from a vector, your primer binding site is located outside the PCR product, so you should get the entire PCR product sequence. Also, vector primers are tried and true. Sometimes, primers you use to amplify PCR products make lousy sequencing primers.
thank you for your reply!
does it means that it is not necessary to do cloning before sequencing? as the problem of missing first 50-70bp can be solved by doing both the forward and reverse primer for sequencing ......
our lab usually done human DNA and we do sequencing directly after PCR (of course after purification step) ......but we haven't done microorganisms......has anyone ever do sequencing directly after PCR in microorganism??
I've been doing Genome Walking with parasite DNA and sequencing the PCR products straight forward (after purifying the band grom gel). It works jus fine provided that you have a amount enough of the product you want to sequence. I have only cloned when the fragment I want to sequence is not easily seen in the gel.
Good luck 
I have no problem sequencing PCR products...
In fact, a case can be made that sequencing PCR products directly is better than sequencing a cloned PCR product, because when you clone a fragment out of the population of PCR products, you may have selected one with an basepair incorporation error, and thus when you sequence this plasmid, you will get back the error (all the plasmid clones will have the error).
But, when you sequence a population of PCR products directly, the product that has an incorrect base in any given position is likely vastly outnumbered by products that have that particular base correct -- thus the sequence called from this population will be correct.
Working on HIV, we do sequence directly from PCR product which works very fine most of the time, we use forward and reverse primers so we get all we need.
But: when we get unclear signal on the electropherogram, due to insertions/deletions (frequently occuring in HIV genome at certain positions) we need to clone and sequence enough clones to account for what we saw on the original electropherogram.
Problem with cloning is: if you have a certain mutation that you would miss with population sequencing (as we call it for direct sequencing of PCR product), it's hard to tell if this mutation is a PCR artefact (no polymerase is error-free!) or indeed a mutation in the original template... And another question raised with cloning is just how much clones would you have to sequence?
so: depending on your needs: do PCR sequencing of cloning.
I seem to recall reading somewhere that three clones (plucked from three seperate PCR reactions) whose sequence all agree was considered required...