Hi Sanny,

If I have understood your question correctly, you have a pair of synthetic primers to clone a GnRH sequence. A band has amplified that is the correct length from several libraries. However, when you sub clone the purified band and sequence it, it is a spurious bacterial sequence.

Providing your primers are specific to the sequence, and the amplification conditions are such that non-specific binding is unlikely, and you have appropriate controls, this shouldn't happen. That being said, many of us reading will have had occasional weird PCR results over the years that cannot be rationally explained, so the only thing I can suggest is asking yourself the usual questions.

How many specific bases of the reading frame from start codon (5') and stop codon (3') did you have in the primers? I usually design them for 16-22bp, with the final pair of bases a CC or GG doublet where possible. If there is scope to extend the original primer sequences by another 4-7bp (again ending in a high temperature doublet) at the 3' end, this may help.

How long is the GnRH sequence - could you try amplifying two halves? (using a pair of overlapping internal primers vs your original primers).

Could the problem be unrelated to your PCR, and instead contamination due to problems with gel purification of the PCR product, digestion of PCR product for cloning, sub cloning of PCR product, or the sequencing itself?

Feel free to add more information about your processes and discuss this here further - it should be easy enough to resolve.

Best, Robin

Hi,

from the title of your question, do I understand it right that you want to create a reporter assay for transcription of the GnRH receptor? If this is the case, you do not need the coding sequence (start to stop) from cDNA, but the transcription promoting sequence elements ('promoter') from the genomic DNA.

What sequence elements are exactly required is frequently unknown, however a good start is usually to clone 1-2 kb upstream from the transcription start site (TSS). Still, frequently there are further regulatory elements downstream in the introns (or even exons), which is why commonly the whole sequence until translation start or even beyond that is included.

On the sequence or cloning problem, it can be tough to amplify sequences from complex templates, which may give unspecific products. However getting a clear sequence from an totally unrelated organism is somewhat peculiar, even if your primer binding is quite unspecific. Is the sequence trace of high quality and does your primer also (at least partially) fit for the bacterial sequence (try primerBlast)? Also, in addition to what Robin has already suggested, consider the possibility that there may have been a mixup (or contamination) of samples and / or sequencing primers at your sequencing provider. I would suggest to start troubleshooting by carefully checking and possibly repeating the sequencing.

Hi,

Ampify using PHUSION., we get 10kb PCR without an issue. Or do two PCR amplifications & fuse using Gibson Assembly.

Thank you

Anand

Dear Robin and Patrick,

Thank you for your suggestions! I'm so sorry for my late reply, I have read your comments just now.

I am using the KOD FX kit. I designed two sets of primers. The first set is designed at the 3' and 5'UTR.

The second set is designed from the start codon for Fw primer (21 bp plus extra TAGA nucleotides, L21 sequence, and Nhe1 cutsite sequence)

and at the stop codon for Rv primer (21 bp plus the Xho1 cutsite sequence).

However, my primers designed in those areas aren't specific to my target gene base on BLAST. I can design a more specific primer set from the Transmembrane domain regions but since in the protocol that I am following for creating a target gene plasmid subcloned into pCDNA vector , i needed to include the whole Open Reading Frame (from start to stop codon).

Currently, I designed three sets of new primers from the untranslated regions and partly the ORF region for Fw primers. Rv primers were designed from untranslated regions only.

As for the transcription promoting sequence elements ('promoter') from the genomic DNA, I still don't have enough idea which part is it in fish GnRH receptor. I will try to research more on this.

Thank you very much!

Sincerely,

Sanny

Thank you Anand for your comment!

I haven't used those kits yet, I'll try reading their protocols.

Cheers!

Sanny

Hi Sanny,

one other thought that has just come to mind - with my commercial head on rather than my geek one. Do you know the sequence of your reading frame (i.e., all of the bases)? If you do, the other alternative would be to generate the reading frame by direct gene synthesis. There are many companies out there who do this, and prices are now extremely competitive pretty well worldwide - to the point where your time performing repeated RT-PCRs and getting the wrong results will exceed the cost of direct synthesis by some margin within a few days. This would also mean that having specified the sequence you can get the restriction sites you need for cloning into pCDNA3.1 - and most companies will also supply the sequence cloned into this vector for a relatively minimal supplement. I'm not trying to discourage your cloning efforts - but if you need the sequence for planned experiments, this may be the quickest way to get there.

Best wishes, Robin

Hello Robin,

Yes I do have the whole sequence already. Thank you! Actually, my senior who is now working at different Institute suggested this to me also yesterday.

I discussed his suggestion to my Associate professor and we might do this if our troubleshooting ideas will still fail. Hehehe!

Thanks again for your help!

Cheers!

Sanny

Hi,

from your remarks, I'm still not sure of what you are trying to construct in the end. pCDNA is a classical expression vector without reporter gene (e.g. luciferase, GFP, ...). Do you want to assess transcription/expression levels or the spatial location of your receptor ('fluorescent tagging')?

In the latter case, the part of your sequence carrying the particular localization signal (signal peptide) should be sufficient (~ 50 aa), usually located at the n-terminus of the translated protein (rarely on the c-terminus), which you would fuse to your reporter protein of choice.

Hello Patrick,

Sorry for the confusion. My target is to determine the binding affinity of my GnRH receptor to its ligand (GnRH). I will be performing Reporter Gene assay using Promega Dual-luciferase report assay system kit. But before that, I have to subclone my whole ORF sequence GnRHR gene into pcDNA3.1 vector.

I did similar study in my previous work DOI: 10.1016/j.ygcen.2017.01.027

Now I am working on different species.

Thanks!

-Sanny