Dear all,
I am working at using ligation-mediated PCR (LM-PCR) to amplify DNA of unknown origin; at the moment I am testing the system using amplicons, which should have the terminal adenine at their 3' termini. I therefore generated dsDNA adapters with protruding thymines so that the matching would be something like this (the protruding nucleotides are in capital):
Upstream adapter
5’-aatccgT-3’ / 3’-ttaggc-5’
Downstream adapters
5’-tctacg-3’ / 3’-Tagatgc-5’
Amplicon
5’-atgcgt…cggtaaA-3’ / 3’-Atacgca…gccatt-5’
The length of the adapters is between 31 and 35 nt, with a GC content between 46.9 and 58.8 percent. The Delta G of the adapters is -70.72 kcal/mole for the upstream adapter and -58.62 kcal/mole for the downstream adapter.
I melted the oligonucleotides in pairs to generate the upstream and downstream adapter with a procedure I found in a publication on LM-PCR: 95 °C for 15 mins followed by 3 cycles of 58 °C for 10 mins and 70 °C for 5 mins. I then used Promega T4 DNA ligase to proceed to the ligation of the adapters to the amplicons that have been properly generated by PCR, which included a final extension step of 90 s. The mix for the ligation contained: 3 µl of T4 ligase 10 ×, 3 µl of acetylated BSA 1:10 [1 mg/mL], 2.5 µl of cDNA [~100 ng/µL], 1 µl of upstream adapter [10 µM], 1 µl of downstream adapter [10 µM], 1 µl of T4 Ligase enzyme [3 U/µL] and water to 30 µl. The reaction was incubated at 15 °C overnight, then the enzyme was inactivated at 70 °C for 10 mins.
However the reaction did not work: on the gel there was only the band corresponding to the original amplicons but no shifted bands that would have indicated the inclusion of the adapters; neither there were bands indicating concatenamers, which I was expecting from random ligation of the DNA. Also the following PCR using primers targeting the adapters did not work.
I used different batches of the T4 ligase, different temperatures of ligation (room temperature or 5 °C) and different amplicons, but there was no sign of ligation.
Would you have some tips on this procedure? Have I got something wrong?
Thank you
I presume you will have either gel or column purified you PCR amplicon and if so the only recommendation I could make is try a adding a little spermidine (around 1 mM) to the ligation instead of BSA
Thank you, I will try the spermidine. The amplicon was in a single and well defined band so I did not purified it -- the background due to the primers would have been removed with the actual LM-PCR and gel purification in the last stage.
Single base pair overhang ligation has low efficiency which may be reduced further with the binding and perhaps ligation of the ATP and TTP carried over from the initial PCR reaction.
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