Sequencing contamination? - (Jul/14/2008 )

Hi,

I've been having a few problems with my sequencing reactions and was hoping for some advice. My aim is to clone and sequence a 1100pb product.

Viral RNA was reverse transcribed, followed by a nested PCR to obtain my desired product.
Due to some non-specific binding by my primers, the product was gel extracted and then purified (this was rerun to check purification was successful).
Following this cloning was carried out using TOPO cloning kits, with a round of plasmid mini-prep.
EcoRI digest confirmed successful cloning (digested product was correct size).
Precipitation of sequencing products was performed by ethanol precipitation 96-well format (I have used this protocol for years & is therefore reliable).
I then submit my plates to my sequencing dept.

On blasting my contigs, the analysis tell me my sequencing product is of human origin (not always the same chromosome, and often a monkey thrown in for good measure). How can I, from pre-cloning, go from a product of the correct size, to post-cloning/sequencing something completely different?

Has anyone had similar problems? Do you think this is a sequencing or cloning problem?

Any comment/suggestions gratefully received,
Bunsen

Hi,
you give the answer of your problem yourself:
Viral RNA was reverse transcribed, followed by a nested PCR to obtain my desired product.
Due to some non-specific binding by my primers, the product was gel extracted and then purified (this was rerun to check purification was successful).

I think the problem is that your "viral" RNA was viral RNA mixed with donor RNA (monkey) and your RT and/or PCR was not specific enough...

So try better primers to increase specificity and maybe do a touchdown pcr.
However most mistakes occur during the RT-step. So use a RT system with proofreading e.g. Transcriptor HiFi (Roche) in combination with gene specific primers (in this case better than random hexamers or oligodT). Then proceed with a HiFi PCR system (e.g. Pwo SuperYield DNA Polymerase) maybe in combination with a touch-down PCR. If you already order at Roche the Transcr. HiFi use the High Pure PCR Cleanup Micro Kit for clean up of your PCR reaction (with or without gel extraction when you only have 1 product). You can elute with very small amounts of elution buffer so you do not need a EtOH precipitation step and save work and material.

It is very unlikely that the mistake occurs after PCR....

Good luck and much success

Thanks for the advice Senior Scientist. I've gone back to the beginning and am re-RTing my RNA using the HiFi kit, so will let you know how I get on. I'm looking at quite a variable section of my virus, so it is very difficult to find specific primers for amplification - the sequencing does work well when it wants to, however it also doesn't as I have described above.

Thanks,
Bunsen