# problem of DNA sequencing - (Mar/18/2008 )

Does anybody here who had experiences of sequencing internal transcribed spacer (ITS) region? The PCR products are not unique sequences for each reaction but has homologous fragments instead. Thus, the results of direct sequencing have double peaks in some positions. How to solve this problem then? Can I seperate these homologous fragments first and then do the sequencing? If yes, what is the proper method? Thanks in advance.

To get the different sequences separately you will need to clone the PCR product and then sequence individual clones. How many clones you will need to sequence will depend on how many different ITS sequences there are. Since you already have a chromatogram of the "pooled" sample, you should be able to estimate the frequencies of the various SNPs in your sample, which should give you some (rough) idea of how many clones you will need to sequence to get at least the most common sequences.

Thanks for your quick reply. But I still can not understand why the sequences can be seperated after they are cloned into T-vector? Based on my understanding, vectors harbouring different ITS sequences could be transformed into the same competent E. coli cell. Recovery of these vectors and then sequencing could cause the same problem as I mentioned in my post (the double peak)? Pls point out the mistake I possibly made during this consideration.

The probability that two vectors will enter the same cell during transformation is not zero, but it's sufficiently low that you need not worry too much about this happening.