DNA degradation? - (Dec/15/2008 )

Hi to everyone

I am trying to ligate blunt end PCR products (pwo polymerase) into ZeroBlunt plasmid, a comercial one for cloning of this type of inserts. As the PCR product are quite big (2-3Kb) the frequency is not too high, but I can obtain some clones. I extract the plasmid and check the good restriction pattern. Then I sequence them, and I find some nucleotides less at the 3' end of my sequence. I have tried several times, and I always find this type of "degradation", but is always different amount of nucleotides dissapearing, from 2, 6, 66, 118 or 200...

I have tried it with different PCR products (as I am doing several clonings in parallel). The PCR was purified with Quiagen from solution, but not from the gel, as it gave a proper and unique band of the right size. The PCR products theirselves were sequenced before cloning into the vector, and were OK.

I think I have any DNAse contamination, maybe in the water I have been using to elute the PCR purification, but, is it a common type of degradation?? Is like there is something "eating" nucleotides at the 3' end of my PCR product..... What do you think?

Thanks

this is most certainly odd.

I have read that DNA polymerase if left at extension temperature and in an environment poor in dNTP can cause 3'-5' nuclease activity. But since your PCR products are okay, it seems that this isn't the problem.

Did you gel purify your PCR amplified DNA? It is possible that these guys are examples of failed PCR products, which won't actually show up in the sequencing as they are in the minority. However during ligation/transformation, they are selected for since they are smaller molecules.

I guess it might be possible that your reagents have been contaminated with a nuclease with 3' activity, but it is certainly strange. I guess to be on the safe side, change all your reagents with new ones.

Oh, which way are you sequencing your ligated insert. If the sequencing primer is too close to the DNA sequence of interest, you will have problems getting good reads. Might this be the problem? It takes at least 20bp (if not a little) from your primer, before you can get a good sequence read.

Brute force and ignorance... given that some inserts suffered only minor damage, can you isolate one which undamaged? You only need one.

Lastly, with current DNA polymerase 2-3kb PCR product is small. Most DNA polymerases can easily amplify 5kb. A big PCR product is above >10 kb. So don't be worried if you have to PCR amplify larger products. Technology is on your side.

this is most certainly odd.

I have read that DNA polymerase if left at extension temperature and in an environment poor in dNTP can cause 3'-5' nuclease activity. But since your PCR products are okay, it seems that this isn't the problem. yES i THINK THE SAME

Did you gel purify your PCR amplified DNA? It is possible that these guys are examples of failed PCR products, which won't actually show up in the sequencing as they are in the minority. However during ligation/transformation, they are selected for since they are smaller molecules. NO. I PURIFIED FROM THE SOLUTION, BECAUSE THE GEL SHOWED A NICE SINGLE BAND. ANYWAY, IF THESE DEGRADATED PRODUCTS ARE THE MINORITY, I SHOULD HAVE FOUND AT LEAST ONE GOOD CLONE!!

I guess it might be possible that your reagents have been contaminated with a nuclease with 3' activity, but it is certainly strange. I guess to be on the safe side, change all your reagents with new ones. I HAVE ALREADY DONE THAT. I'LL KNOW BY THE END OF THE WEEK....

Oh, which way are you sequencing your ligated insert. If the sequencing primer is too close to the DNA sequence of interest, you will have problems getting good reads. Might this be the problem? It takes at least 20bp (if not a little) from your primer, before you can get a good sequence read. YES, THE SEQUENCE IS LIKE 400 BASE PAIRS FAR FROM THE PRIMER, AND IT CONTINUES CLEARLY WITH THE PLASMID BACKBONE. THE SEQUENCE ITSELF IS CLEAR AND GOOD.

Brute force and ignorance... given that some inserts suffered only minor damage, can you isolate one which undamaged? You only need one. I KNOW, BUT I HAVEN'T FOUND ONE YET

Lastly, with current DNA polymerase 2-3kb PCR product is small. Most DNA polymerases can easily amplify 5kb. A big PCR product is above >10 kb. So don't be worried if you have to PCR amplify larger products. Technology is on your side.