Dear Mrs. Huijsers, Thank you for your question. If you would like to determine the length of your amplicon using your primer, you can utilize a program or method called Primer Map, which is one of the features available on bioinformatics.org. This open-access website allows you to simply submit your targeted gene and primer sequences to predict the amplicon length. Best regards, [Antama S.]

What organism are you targeting in your research/project, Mrs. Huijsers?

CTP2-CP4-epsps is a target gene from the bacteria Agrobacterium tumefaciens used for GMO detection. It is a gene construct that has CTP2 and CP4-epsps combined and it is used in a lot of GMO's, therefore used in GMO detection.

Hi Kaylee,

I think I can provide you with your amplicon sequence. As a CTP-CP4-epsps is a synthetic sequence which has most likely a patent on it I searched NCBI blast for "patent sequences" - here I got several hits for your primers - one of them was related to soybean event MON89788 (and several other patents). In this sequence you will find your primer sequences as well as the probe sequence. The amplicon is 139bp in size. I also found your exact qPCR assay (same primer and probe)- here it is also mentioned that the amplicon is 139 bp in size. I attach the sequnce data ( as word document and as Ape document as well as the data regarding the qPCR as well as a picture of a blast with the 139 bp amplicon. I hope this will help

Thank you so much for your response! It really helped me.

I have another question for you if you dont mind.

I have found my amplicon for my gene construct P-35S / nptII (another GMO target in my analysis), which is:

TATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTCGAGTATAAGAGCTCATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAATTACATTTACAATTATCGATACAATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATC

These are my oligo's:

• P-35S/nptII-F 5′- TAT CCT TCG CAA GAC CCT TCC -3′
• P-35S/nptII-R 5′- GAT TGT CTG TTG TGC CCA GTC A -3′
• P-35S/nptII-P 5′-Cy5 - AGC CGA ATA GCC TCT CCA CCC AAG C -BHQ2-3′

Now, I can find my primers, but my probe is nowhere to be found in this amplicon sequence. My PCR analysis works, so there has to be an amplicon sequence where these primers are used together with this probe. My reference used for the analysis is MON1445.

I got the amplicon sequence from: https://www.addgene.org/160646/sequences/

Can you maybe also help me to find the correct amplicon sequence for P-35S / nptII?

Do you maybe also know the answer to my previous asked question:

• Is it okay to add the amplicon sequences for all my targets together in a single synthetic template, or should I include spacer sequences between them? And if so, what should the spacer sequences be?

Thank you very much for your help!

Hi Kaylee,

please have a look at the following links:

https://euginius.eu/euginius/pages/gmo_detail.jsf;jsessionid=6Uap8b8qJDV56yejMGHk_dzYeJ2x53BFV1hZKASv.subs262?gmoname=MON1445

and

https://euginius.eu/euginius/pages/method_detailview.jsf?method=QL-CON-35S-F%2FnptII-R

here you will find your exact assay including the amplicon sequence.

Regarding your second question about the synthetic template I am not sure about this as I have never done multiplex PCR myself, however you might want to check out the pdf you can find under this link:

https://gmo-crl.jrc.ec.europa.eu/doc/KJNA30708ENN.en.pdf

I hope this is helpful.

Interested in your project as I work at Wageningen University at GMO detection.

Have a look at our website EUGINIUS website, it contains a lot of information on GMO detection