The gene I am working on has 6 copies. I have retrieved genomic DNA sequences of all of them from public domain. I also managed to get FL-cDNA of 5 of them. The missing one (I call it A-2) has an in frame stop codon. I developed a specific PCR to amplify 3’end and manage to get RT-PCR amplification of the 3’ end of A-2, but not FL-cDNA. The primers I used to amplify FL-cDNA works because they amplify genomic DNA. My cDNA samples are also OK as I used the same samples to get the other copies amplified.
One possibility is the transcript is low copy numbered. Can you give me an advice, how may I proceed?
What is the expected size of the A-2 transcript? Are you trying to clone it for purposes of forced expression, or are you trying to assay endogenous transcript levels by qPCR?
Hi Chandima,
Just to see if I understand you correctly, you are trying the amplify the full length of the A-2 variant, but you only get amplification using primers you made for the 3' end of the gene, is this correct?
You can use the first pcr product as cDNA and use this for the second pcr . Then you will have the copy of the copy. You can try like this about 4 times and reach an enriched copy number
. Dear all
Thank you for the comments. I am trying to get the FL-cDNA to get it sequenced. I did get amplification of a short fragment (195 bp) using primers on the 3' prime end. So the geneis probably expressed but no amplification for FL-cDNA. Burcin, if I use the 1st product as cDNA would you sugget diluting the PCR product?
Best regards
Chandima
Since you have just 6 copies for the first run, the dilution is not needed for the second. But you should always keep in mind the gained copy number.after about 5000 copies, the you can make 1:10 dilution..hope it helps..
Just to add to what Burçin said, I don't know how many PCR cycles you usually run but I would suggesting running no less than 40. As for the second PCR run, 2 to 4 ul of undiluted product can be used as template. If that still doesn't work, I recommend trying a one-step RT-PCR kit (if you haven't already). They often provide better sensitivity than two step kits.
I am assuming that the mRNA sequence of this isoform is known. If so, are you sure that there is only one splice variant for A2? If there are other variants, probably your primers for 5' end does not correspond to the "unknown" variant. You may want to consider doing a 3' RACE.
I don't think you mean there are 6 molecules of transcript in your extract, but rather 6 transcript variants each with different abundances. Am I correctly interpreting your post?
Since your 3' primer appears to be functional, and your 5' primer works on the other gene variants, might you not infer that A-2 has a different transcriptional start site or is spliced in a manner that eliminates the homology to your 5' primer? You may need a different 5' primer to amplify the FL cDNA for the A-2 variant.
Dear Daniel
We thought of that possibility, but the problem is how to find that? how to design 5 primers for the alternative site? Can you think of any way to do that other than constructing cDNA libries
Thank you
cheer
chandima
Depending on the species you are working on, there may be publicly available data on mRNAs that could help you design your oligos. What is your target species? Are there pre-existing cDNA libraries available that you could conceivably screen?
Are you certain that your 3' primers amplify cDNA and not genomic DNA? How do you know the A-2 variant is expressed?
Hi
The specific PCR I developed to amplify 3' end of the gene (product size 195 bp) works on both cDNA and genomic DNA so it may be possible the gene is expressed. But I could not amplify the FL-cDNA.
cheer
chandima
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