Make construct so it is only thing that can PCR after transfection - (Mar/10/2009 )

Hi everyone,

I'm hoping for some inspiration. I am planning on cloning a dna fragment which contains three exons and the two introns in-between them. I'm also including around 100bp upstream of the first exon and downstream of the last exon.

I will be transfecting into cells in culture and then purifying RNA and running PCR to look at how the mRNA was spliced (# and order of exons). But because the construct will be from human DNA and going into human cells, I would not be able to isolate the products formed by my transfected construct.

Is there a way for me to add 20-30bp of unique sequence to the beginning of the first exon prior to cloning?

I am planning on using the Clontech In-Fusion kit which requires special primers that have 15bp overlap of vector sequence...and so this primer must go at the beginning and ends of the extra introns.

I was thinking of maybe just using the primer to create a small "fake" exon (maybe 15bp)...which should work as long as I start it with AG and end it with GT. And then I could target the primer to mostly this sequence...and should gain specificity.

Sure it will work. Such method is very commonly used to differentiate exogenous from endogenous sequences and is known as adding barcodes.

Thanks for the reply!

Would it be better to create the "fake exon" or just add on a section to the first real exon? If I create the "fake exon", I would be cloning two inserts (vector--fake exon--my real insert--vector). Adding a section to the first real exon means I have to clone three inserts (vector--intron upstream of exon 1--additional section of exon 1--my real insert--vector).

Would the second approach make things easier since I don't have to worry about how a new exon might be spliced?

Since the In-Fusion kit is PCR-based, I might have to actually use PCR to generate the unique segment. So I guess I can just amplify a 50bp region of some nonconserved intron on a different chromosome.

Thanks for the suggestions