Digest-Ligase-PCR Problems (T-DNA fingerprinting gone wrong) - After ligating digested product and doing PCR on it i get no DNA smear (Oct/01/2007 )

Hi,

I am doing T-DNA fingerprinting and I ran into some problems. I take some plant genomic DNA, digest it completely using a tetracutter (in this case MseI) then I am to ligate it with adaptor ends (sticky end for MseI digestion product). After that, I will PCR the ligated DNA with a primer based on the adaptor end. Sounds simple and straight forward but unfortunately I seem to be stuck here.

The digestion was done for 5 hours with MseI and running 1 sample on agarose shows even smearing as expected so I don't think there is a problem here.

Ligation was done with adaptor DNA : 2 hours ligation (Room temp) and 2 day (4 C).

Ligated product was PCR amplified using the lowest possible annealing temperature -- 37 C which is much lower than the primer's calculated Tm of 48 C (specificity isn't important at this point, just getting the ligated products amplified is enough). The total digested DNA was used ( minimum 20 ug -- enough to see the smear pattern on gel)

After the PCR i took 1 tube and ran all of it on gel to see if I get the expected smear which looks just like the complete digestion only brighter (as the PCR will amplify all the digested fragments which can ligate with the adaptors) Results shows no DNA , not even smearing.

Did ligation eat up my DNA ? If the ligation and the PCR failed I still expected to see the digested DNA smear but I get zilch, nada , zip , nothing Anyone knows how I can sucessfully digest with MseI, ligate the sticky ends with the adaptor and then PCR the whole thing to get amplified smears ?

If it helps, when/if I ever get the darned smear I will reamplify with Biotin labelled gene specific (T-DNA specific) primers and the adaptor based primer and use the straptravidin-magnetic method to separate the DNA which has the T-DNA from the countless DNA fragments without the T-DNA. This method is based on :

Journal Title - Euphytica
Article Title - Identification and characterization of T-DNA inserts by T-DNA fingerprinting
Volume - Volume 123
Issue - 1
First Page - 75
Last Page - 84
Issue Cover Date - 2002-01-01

Author - I. Theuns
Author - P. Windels
Author - S. De Buck
Author - A. Depicker
Author - E. Van Bockstaele
Author - M. De LooseDOI - 10.1023/A:1014415619527
Link - http://www.springerlink.com/content/551150d541uepj63

Repeated this a few times but results stay the same.

Other info that may help in the troubleshooting :

1) My digested DNA was kept in the fridge for a few days but I checked if the DNA was still there just before ligating , it seemed ok on gel .

2) I did not do heat inactivation of the MseI after digesting, just chucked it in the fridge. Normally I do this and don't have a problem with it.

3) I couldn't check the ligated DNA on agarose since, for fingerprinting the T-DNA I have to PCR the whole total genomic DNA

4) Yes, there was EtBr on the gel , the ladder turned out perfectly

5) Since I used the whole contents of the eppy tubes, no worries on not mixing / resuspending the DNA when used as PCR template. (I use the original eppy tubes that the ligation and digestion was done in .. to avoid losing those perisistent DNA sticking to the eppy wall)


Any advice/explanation/help please ? My supervisor is hounding me .. help !

p.s : Pretty please , with sugar on top

Your supervisor should be helping you not hounding you. Several things come to mind. Where did the adapter come from? Is it phosphorylated? It must be. Heat killing the enzyme is essential unless the adapter ligation fails to recreate the site. It sounds as if you are add FAR too much DNA to the PCR reaction as a template. Less is more. Try again with 20 ng not 20 ug. I have never had a PCR reaction work when the annealing temperature was less than about 49 degrees. Choose a temperature 5 degrees below the Tm of your primer. Design the primer to have a Tm of 60 degrees. Run your annealing temperature at 55 degrees, it almost always works. If you have a gene specific primer for the second stage, why are you not using it for the first PCR, which would provide much more specific products, and perhaps avoid the need for fishing the correct results out of a mix.

You are right about the phosphorilation , didn't pay any attention to that .. my bad.

As for the DNA quantity, each tube represents genomic DNA from a transformed leaf disc containing GUS expression spots and the reason for doing the PCR is to determine the amount of the inserted copy number during transformation so it's essential that all the DNA recovered from the leaf disc be used for PCR. The idea is to use PCR to amplify any digested fragments containing the gene inserted into the plant so we can determine the copy number and isolate the flanking region of genomic DNA at the upstream location from the transformation insertion point, if I use a portion of the total genomic DNA then I might miss some fragments carrying the GUS gene since not all cells in the leaf disc has the same insertion point and copy numbers (chimeric transformation is a very high probability with this method)

I was thinking perhaps I can aliquote each genomic DNA tubes into multiple tubes and do PCR on them and pool the resulting amplification results into a tube. That would allow me to reduce the amount of DNA used but still not miss out on any intergration events.

I totally agree with you on the Tm and using the gene specific primer and are changing my PCR temperature to original primer Tm - 5 C which is 52 C. I have no idea why I used such a low Tm .. probably got into the " lower temp = more products since primer will stick to anything it likes" mindset , thanks for correcting me.

Thank you very much on your suggestions. Gives me lots of places to check and troubleshoot. Really appreciate it. I'll post here on how things work out. Peace