Hello,

if you have sequence information about the orthologs in the target organism, just align the two sequences and look for regions of variation. They would be the perfect targets for primer design. This sounds obvious, I guess.

If you do not have sequence information, then I would rely on GenBank. Extract all sequences from orthologs, run a multiple sequence alignment and look for regions of high variability. They are likely to be different in your target organism as well because they evolve faster than the rest of the sequence. Next step: just design the primers and test them before transformation. If they work with the donor but not with the target, voila, that's it.

Good luck,

Ralf

If you are transforming a target organism, isn't it obvious that you will only detect transformed gene in target only upon successful transformation?

As Abdur and Abhijeet said, designing specific primers to a specific known sequence gene is nowadays easier than cooking noodles. The matter is how this recombination would work if the target does not share any sequence identity/similarity to the gene transformed. You might end with transformants those gene was recombined somewhere in the genome.

Thank you for your answer Mr. Abhijeet Singh. The problem is, the target gene has some orthologous genes in the plant genome which I want to transform (so the primers I design has complementary regions in the target plant so it's not specific for my transgene only). And I can't find all the orthologues. If i had the sequences of all the orthologous genes then I could have used multiple allignment tool to design the primer manually. I hope you understand what my problem is.

Thank you Mr. Md. Abdur Rahim for the answer.

The reason I need the gene specific primer is to confirm the transformation process's success. But can you please tell me why is it important to design a primer which will cover the entire sequence?

Hello,

if you have sequence information about the orthologs in the target organism, just align the two sequences and look for regions of variation. They would be the perfect targets for primer design. This sounds obvious, I guess.

If you do not have sequence information, then I would rely on GenBank. Extract all sequences from orthologs, run a multiple sequence alignment and look for regions of high variability. They are likely to be different in your target organism as well because they evolve faster than the rest of the sequence. Next step: just design the primers and test them before transformation. If they work with the donor but not with the target, voila, that's it.

Good luck,

Ralf

Thank you Miss Loubna Youssar (@Loubna Youssar) for your words. I know it is simple and easy but the problem is that the target gene has homologous regions in the genome of the target plant (probably the orthologous genes). So the designed primers have complementary regions in the target plant's genome. So if I use those primers, I will get positive results even though the transformation doesn't take place. And another problem is, I am unable to obtain the sequences of the orthologous genes. If you can tell me any ways to identify the orthologous genes in the genome of the target plant I would be grateful. Will ensemble (https://www.ensembl.org/) work?

Thank you very much Mr. Ralf Koebnik for the info.

Actually the question is little confusing when you mentioned "I want to design a specific primer for that gene which will not share any sequence identity with the target (the plant I want to transform)" Talking about orthologuos genes, these last have to share 70% identity and at least 50 % of the sequence length.

First of all, do you have the genome sequence?

Miss Loubna Youssar , I have the sequence of my target gene (one inserted into the construct) but I don't have the sequences of the orthologous genes (Or genes which have the primer (primers which I initially designed using Primer 3) complementary regions).

Mr. Md Abdur Rahim , I already have the construct. So I don't need to clone the target gene. I am transforming the plant for changing some physiological conditions for stress tolerance. But I am designing the primer to assay the transformation process.

Hi Md Fahmid Hossain Bhuiyan!

Its been a nice question and I think I can help.

But I need to know if you have the orthologous sequences (in the plant) of your target gene.

If you have, then you just need to find the unique portion in your construct sequence and design primers which would bind to those portions. In case you already have targeted the unique segments of your construct and those are discursive for designing a primer set, then I think you need to take the help of multiplexing.

Since PCR primers are cheap relative to the amount of time it could take you to solve this problem analytically (and you don't even have the information for an analytical approach), I would suggest just trying various combinations of primers on the DNA of the target plant. When you have a pair that works on your construct and does not give a product from the target plant, you can use that pair to test successful transformation. You could try to find a single pair for the whole construct gene or if necessary, two or three pairs for overlapping parts of the inserted gene. When you test the candidate pairs, I would suggest testing several PCR conditions with different annealing temperatures. Then choose the PCR condition with the highest annealing temp that still works well with your construct--just to make sure that there is a minimum chance of interference from any highly similar orthologs in the target plant. Also, to best simulate the conditions of your planned test, you can also just mix an appropriate amount of your construct plasmid with DNA of the target plant. Adjust the concentration of the target plasmid so that it matches what it would be as a single insert in the target plant genome. When you find a primer pair that produces a good product from this complex mixture but NOT from the target plant DNA alone, you can be pretty sure that it is a reliable signal for successful transformation.