coligation of PCR amplicon

[amhro]

Does anyone have a protocol for coligation of PCR amplicon for sequencing on the Ilumina platform. As I understand the way to proceed with library prep for PCR amplicons is coligation, fragmetation, gel fraction and then adaptor ligation is this the best way to do it or is there any easier way? We have pools of PCR product between 300-450bp that we want to sequence.

[berthold]

Hi,

did you get an answer to this last year? I'd be interested in such a protocol as well. We actually did the experiment through GATC in Germany some time ago (they did all the steps in-house). It worked fine, but you need to apply appropriate bioinformatics in order to "restrict" the co-ligated fragments on the computer. This can be complicated if different primer sets are involved.

[bbeitzel]

I routinely do this for sequencing viral genomes out of complex mixtures. I PCR ~6-10 amplicons spanning the genome (12kb) with ~300-500 bp overlap between the amplicons. I then mix the amplicons in equimolar ratios (shooting for 5-10ug DNA total) and do a column PCR clean-up. I generally elute this into 25ul and then EtOH precipitate to get a pellet. I resuspend the pellet in 3.5 ul H2O, and then add 0.5ul 10X T4 ligase buffer, 0.5ul T4 polynucleotide kinase, and 0.5ul T4 DNA ligase, and let the mix incubate at RT for 4 hours to overnight. This will generate high molecular weight DNA, which I then fragment and use for library prep.

Some notes:

1) I use Phusion polymerase, which generates blunt ends. If you use Taq, you'll have to polish the ends to get rid of the A overhang.

2) I have found that the smaller the ligation volume the better. If I tried to ligate in higher volumes (25ul or more), fewer of the amplicons get converted to HMW DNA. I don't know if you could get a similar effect in a larger volume by adding PEG.

3) If you use primers that are phosphorylated, you can omit the PNK in the ligation reaction.

4) The DNA pellet does not resuspend very well in 1x T4 ligase buffer, which is why I resuspend in water and then add buffer.

5) I have also run a modification of this protocol by adding a unique restriction site to my PCR primers (AscI in my case,) digesting the PCR product with the enzyme to generate sticky ends, and then ligating. This seemed to work a little better then PNK, but adds an extra digestion step. You also have to do a column cleanup of the digest to get rid of the small pieces of DNA that get trimmed off of the ends of the amplicons. If you leave them in during ligation, they can block ligation into HMW DNA.

Hope this helps,
Brett

[berthold]

thanks Brett, that helps extraordinarily well
will make my student very happy
best regards,
Berthold