Proving Existence of Introns!

[BioGradStudent]

Using today's technology, how would a person prove the existence of introns starting with the cDNA of B-globin?

I was thinking about using a microarray approach. Have oligos from the chromosomal gene on the slide and hybridize the cDNA to it. If introns do exist, then there should be some spots on the slide that are not hybridized.

Am I on the right track here? Is there a better way to do this?

Thanks!

[GreenDog]

At firs I had four suggestions, I wrote them but it all got erased, which is a good thing because only one of them was right.

The question implies you only have the cDNA and not the genomic sequence, so the microarray doesn't fit the question.

The only thing I can see is PCR. Use the cDNA to plan many primers (20 maybe), and use them on cell extract. Some will amplify what you thought they should, but some will amplify very large fragments. Sequence them and you'll get the introns.

[BioGradStudent]

Thanks, I was starting to think along the same lines!

[MrMistery]

Here is a very fun way. Black is your cDNA, red is radioactively labeled chromosomal DNA containing the gene for Beta globin. You first hybridize them. then treat with either S1 nuclease or exonuclease VII.
S1 nuclease is an enzyme that degrades all single stranded DNA, exonuclease VII only degrades single stranded DNA that has a free (either a 5' or 3') end. As you can see, this experiment clearly shows that the region termed b is not included in the mRNA. It does not tell you how long the intron is, but it does prove the existence of introns.
The picture is from an MCB 52 lecture in fall 2008, Harvard College.

[GreenDog]

Radioactive but nice.