In allotetraploid B.napus plant transcriptome, Based on pacBio 3rd generation sequencing, one gene has evidence of Alternative splicing (AS) in it. This gene has four exons and produces 2 distinct transcripts. The structural analysis of two transcripts shows that transcript 1 is produced by using only exon 1 and 2 of gene, whereas, the transcript 2 is produced from exon 3 and 4. Therefore, these two transcripts have no overlapping sequences between them. I have to verify the presence of these two transcripts. Is there any other other strategy to amplify the these two transcripts at once and show the evidence that these two transcripts are definitely transcribed from the specific gene, except using the transcript specific primer for each transcript (using transcript specific primer will just help me detect that transcript but will not provide enough evidence that it is originating from this specific gene.)?
- This should not be hard as the exons you mentioned has clear separation and designing primers for the specific primer sets based on the alternative splicing could be an easy solution
- However, I am not sure, what specifically you mean by amplify. If you are talking about DNA/genomic level, then I doubt if this can be done. But if you are talking about cDNA, then you could be able to do that.
- Regarding your confusion, you will be having the sequence of the gene and transcript and it will be obvious that transcript is originating from that gene, so I guess that is not a problem.
Do you have evidence that these transcripts are alternatively spliced from the same pre-mRNA and are not coming from two different genes? For example, are there other splice isoforms with combinations of exons that include 1 or 2 with 3 or 4? Do you see evidence of the full length (unspliced) pre-mRNA including exons 1-4 in the PacBio reads?
As Abhijeet Singh mentions, even if you use transcript-specific primers for PCR amplification, you can Sanger sequence some of the products to be certain that they come from the gene that you're expecting.
You have to design 2 sets of transcripts specific primers i.e. Primer set 1 including exon 1& 2 for transcript 1 and Primer set 2 including exon 3&4. Use the multiplex PCR to amplify both in a single reaction.
For multiplex PCR prepare the primers as nearby Tm.
I think it will help you more.
Hi Sehrish Sarfraz ,
I come a bit late but maybe this could be useful for others.
You totally can amplify both transcripts with the same set of primer.
For this, you need to design a forward primer targeting the 5'UTR of your pre-messenger RNA, and the reverse targeting the 3'UTR. If, as you said, both your transcripts are spliced from the same pre messenger RNA they have to share those features.
Run the PCR products on an agarose gel.
Assuming that both your transcripts isoforms don't have the same lenght, you can extract both band with a gel cleaning kit and then send them for SANGER sequencing as a verification.
Hope it helps,
Philippe.