Proving Existence of Introns!

Genetics as it applies to evolution, molecular biology, and medical aspects.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
BioGradStudent
Garter
Garter
Posts: 11
Joined: Mon Sep 08, 2008 6:56 pm

Proving Existence of Introns!

Post by BioGradStudent » Sun Jan 11, 2009 11:47 pm

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!

User avatar
GreenDog
Coral
Coral
Posts: 126
Joined: Sun Mar 27, 2005 8:37 am

Post by GreenDog » Mon Jan 12, 2009 4:53 pm

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.
"When In Danger Or In Doubt Run In Circles Scream And Shout"
Lawrence J. Peter

BioGradStudent
Garter
Garter
Posts: 11
Joined: Mon Sep 08, 2008 6:56 pm

Re: Proving Existence of Introns!

Post by BioGradStudent » Tue Jan 13, 2009 6:49 pm

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

User avatar
MrMistery
Inland Taipan
Inland Taipan
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)
Contact:

Re: Proving Existence of Introns!

Post by MrMistery » Thu Jan 22, 2009 2:48 am

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.
Attachments
proving introns.jpg
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

User avatar
GreenDog
Coral
Coral
Posts: 126
Joined: Sun Mar 27, 2005 8:37 am

Post by GreenDog » Thu Jan 22, 2009 6:02 pm

Radioactive but nice.
"When In Danger Or In Doubt Run In Circles Scream And Shout"
Lawrence J. Peter

Post Reply

Who is online

Users browsing this forum: No registered users and 6 guests