2 replies to this topic

[ah6tyfour]

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.

[pcrman]

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

[ah6tyfour]

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