I have been trying for a long period to sequence over a region in the promoter of a gene of maize where a transposon is annotated. In some genotypes the repetitive sequence (TAx) is rather short (70 bp) and we succeeded in sequencing. However, some genotypes are really challenging and we don't even get a PCR product. What we have tried so far:
* primer optimization, we are very close to the annotated transposon, the prime positions are about 50 bp up- and downstream of the flanking sites
* different polymerases: standard Taq, KAPA HiFi, Phusion, HotStar HiFidelity, Velocity
* increased elongation times, up to 10 min because we don't know the actual size of the repetitive element (I expect it to be rather long, several kb)
* lower elongation temperature because of TA rich sequence
* + up to 5% DMSO
If anyone has anadvice, please let me know, I am grateful for any hint!
Svenja
Hi Svenja,
I can not promise anything because I have no specific experience with repetitive sequences. However, for DNA's where nothing works, we were often successful by using the FailSafe buffers from Epicentre (supplied by Biozym in Germany).
http://www.epibio.com/applications/end-point-pcr/end-point-pcr-kits/failsafe-pcr-system?details
They provide 12 different buffer systems for difficult templates and often one or two of them worked for us. I have never asked why.
Good luck!.
Hi,
Infact as suggested by Uwe Borgmeyer above, Roche also used to provide a PCR optimization kit.
Why don't you do a southern blot of the genotypes (using a repeat motif probe) to guess the possible length heterogeneity of that region. Then an appropriate PCR assay could be designed.
See if you can get some information from the contigs available in the Maize genome database
bye
A couple of notes/suggestions:
1) GoTaq Long (by Promega) has worked well for me with PCR or repetitive sequences. Mine were longer direct repeats and only ~1kb total, but that Taq still worked better than others that I tried with.
2) Depending on the type and length of repeat you may be getting stable secondary structures that need to be dealt with, potentially with longer or higher denaturing steps.
3) Use ‘nested semi-random’ PCR with a single primer specific to one side and an ‘arbitrary’ primer until you get a product (several methods can be found for this).
4) Are you even sure that repeat is there in the strains that you are looking for that don’t PCR? If basing of a genome assembly and the repeat is found in other locations of the genome this could actual be an assembly artifact if it is not present in all strains (just a thought when dealing with mobile elements....it would have excised with you primer site, or you may have gotten another Tn element to insert into the original one messing up your primer sites that way)
Thanks to all of you for your suggestions, I will definetely try the FailSafe Kit and modify the denaturating step.
Will keep you updated!
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