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I think the explanation is this:
The source is natural viral RNA which is a mixture of naturally mutated sequences (e.g. flu forms such a quasispecies)
See:
http://www.virology.ws/2009/05/11/the-quasispecies-concept/

The pooled RNA has an average sequence that you see when you sequence the pooled cDNA (individual mutations are hidden by the averaging effect of having many sequences present).

But when you clonally separate DNA molecules by transformation (1 plasmid enters 1 cell to yield 1 colony), you see each individual molecule represented 100% in the sequencing chromatogram from the plasmid DNA that you have isolated from colonies.

This is effect is commonly observed when sequencing influenza virus isolates from patients. It will have nothing to do with the E. coli strain. You can avoid it completely by using gene synthesis.

Darren


2012/1/24 Rubén Sánchez Eugenia <[log in to unmask]>
Dear everyone,

I am trying to clone a viral protein in the E. Coli BSJ strain and i am having some problems.

I start from the viral RNA carrying out a reverse transcription and PCR (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for mutations, there are no mutations. So the RT-PCR works fine.

Then, I digest the cDNA and I ligate it with a pET plasmid to transform the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR) but when I sequence them I get various mutations (aprox. 2 miss-sense) on the inserted cDNA. Furthermore, these mutations are different among different transformations and even among colonies of the same plate (in the same transformation).

Maybe these mutations are produced by the cell (because of the lack of mutations in the cDNA) but these E. Coli clonning strains are supposed to be "optimized" to prevent the insertion of mutations. So I have no idea about what may be the problem.

I hope you could help me. Thank you.

Best regards,

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Rubén Sánchez





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Dr. Darren Hart,
Team Leader
High Throughput Protein Lab
Grenoble Outstation
European Molecular Biology Laboratory (EMBL)
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