t-dna insertion checking - (May/27/2007 )

hi everyone
I want to check how many t-dna inserions do i have in my mutant (arabidopsis).
For that I thought of southern blot and now, I need to do a probe....
if I use one set of primers to check the insertion, they will only produne one band on the gel...it doesn't matter how many copies of t-DNA there are in the genome .....But many people is doing it like this.....can any body explain me how does it reallly work.

thanks in advance

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Hi solmaniar!
It doesn't depend on the probe, it depends on the cuts made by your restriction enzyme.
Southern blot starts with the digestion of your genomic DNA: that's crucial to choose the right enzyme.
You make the restriction map of your T-DNA and you see that your enzyme cuts only 1 time in a site external from your probe. In other words, the enxyme finds its site, it makes its cut producing fragments.
As you have a single cutting site in your T-DNA, every cut will produce new fragments of different lenght.
As they have different lenghts they will separe during electrophoresis, so the probe will detect them in different points of the course producing multiple bands.
I hope I have not confused you more...

Let's complicate this a bit more: if your enxyme cuts into your probe sequence, you will find n+1 bands, where n is the number of insertions.

It all depends on how you design digestion, then on how you desing your probe.
I hope I was good enough in explaining, otherwise I hope I will have the chance to try again.
Don't hesitate to ask!
Cheers!
ILA

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Hi ILA!

Thanks for your answer!!!
Let me see if I understood.... So, I make sure to have only one restriction per enzyme within the t-DNA insertion...and no restriction sites within the probe. To check for different lenght bands I need to cut the same sample with all the enzymes that are in the t-DNA sequence.... all the nezymes to be sure?

By any chance.....does it have anything to do with the RFLP technique?

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Dear Solmaniar,

first of all, let me clear one thing: you cut with a single enzyme, not with every enzyme that has a site in the T-DNA.

You are quite right: in a certain sense it's the same concept at the base of RFLP, but in this case the polymorphism is caused by the presence of insertion(s).

Step by step:
You use a software to get the restriction map of your T-DNA, then you choose an enzyme that cuts only once.
Now you know the position in your T-DNA of the restriction site and the probe.
Let's say that the enzyme cuts out of the probe, so it will divide the T-DNA in two halves and the probe will recognize its target sequence in one them.
Now consider that, you don’t know where, but there will be other restriction sites somewhere in your genomic DNA.

You make your digestion, you let your genomic DNA fragments separate by electrophoresis and then proceed with membrane transfer and hybridisation.

If you have a single insertion, you have a single target sequence for your probe and that will be located in the fragment starting from your cutting site.
No matter how many times your enzyme will cut your genomic DNA again, since those fragments won’t be recognized by the probe.

If you have multiple insertions, you expect to find more bands with different heights.
Your enzyme will cut always in the same site inside the integrated T-DNAs. WHat will be different are the cuts it makes in the genomic DNA downstream your insertion. They will be different depending on where the insertion has integrated in the whole genome.
You can’t predict how long the fragments will be, but you know that they will be polymorphic and that everyone of them will contain a whole hybridisation sequence. The result is a pattern of bands.

I hope my explanation has been a bit better this time…
Let me know!
ILA

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Thank you very much ILA!!!! I think I got it right now....I don't like starting experiments if I can not figure before the whole process in my head.
I think I will finish within a week...

Thanks again

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Anytime!

It takes me 4-5 days to perform the whole analysis, so I suggest you to be careful and take your time. If I'm not wrong, this is my time-table:
1st DAY – o.n. digestion of samples
2nd DAY – gel electrophoresis, depurination and denaturation washes, over night transfer of DNA to membrane
3rd DAY – DNA fixing to membrane, prehybridisation, over night hybridisation
4th DAY – stringency washes, Digoxygenin labeling, membrane o.n. exposure to photographic film
5th DAY - film development (I hope "development" is the right word... )

Good luck!!!
ILA

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