How to determine 3' end sequence of bacterial gene - (Jan/17/2007 )

Hi,

I would like to determine the 5' and 3' end of a bacterial gene for which I only have the internal sequence. I understand there are kits based on the RACE method, but I'm not sure if these can be used for bacterial genes which don't have polyA tails.

Could anyone suggest what I can do? Thanks very much in advance.

Victor

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hi, hong kong guy,
actually, I don't understand the question.....if you have the internal sequence, and why don't you send the genomic DNA to some company and tell them make the primer base on the internal sequence for sequencing ?

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Are you attempting to find the genomic DNA sequence, or are you trying to find the transcription start and transcription stop locations, when you have the genomic sequence. You would use quite different techniques to answer these questions.

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Hi,

Do you mean sequencing directly using the genomic DNA as template? We've tried that, but somehow could not get it to work. It might be because the organism is supposedly high GC (~70%). It's an actinomycete.

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Hi,

Currently, we only have the sequence for an internal fragment of the gene. Based on homology with known sequences, it would appear we have a major fragment of the gene and are close to the transcription start and stop locations. We would like to determine the sequences up to the transcription start and stop sites, and if possible, also the sequences flanking the start and stop sites.

What would you suggest? Thanks!

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if you have the internal sequence and an expected gene size you could try inverse pcr from a genomic library, i have used it to pull out a gene from streptomyces.


hope this helps!

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You could also look at the vectorette idea to capture as much sequence 3' to your gene.
vectorette

I hear it is better then inverse PCR for similar applications.

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