T4 PNK for phosphorylation of oligos

[sindhu_kuttan]

The oligos we give for synthesis have OH groups at both 5' and 3' ends. which means a forward primer can be used as a reverse primer too. So if I have to 5'-phosphorylate a forward primer using T4 PNK, I wonder how would it identify the 5' end when both ends are hydroxylated?

[mchlbrmn]

"which means a forward primer can be used as a reverse primer too."
No, even without the phosphate the nucleoside is not symmetrical. The #5 carbon of the ribose hangs off of the circle, with the number 4 carbon being linked back to number one. The three dimensional shape of the ribose is not symmetrical, and then there is also the big base hanging off of it, in a specific direction. All of this has a specific shape that enzymes must fit onto. (Actually, my memory from school is getting fuzzy, but I think this is accurate.)

[relaxin]

I agree. The OH-groups at 5' and 3' are in different conformations. PNK adds phosphate only on the 5'-OH, while DNA polymerase can add another nucleotide only at the 3"-OH. The DNA chain elongation always goes from 5' to 3" direction. So the forward primer cannot be used as reverse primer. The reverse primer sequence has to be reverse complimentary of the forward primer.

[sindhu_kuttan]

So the forward primer cannot be used as reverse primer. The reverse primer sequence has to be reverse complimentary of the forward primer.

Well, I was hunting for a particular gene and sequenced a few of the TAIL PCR products, only to find the forward primer sequence at both the ends! It was a non-specific amplification for one, and I thought I could look for a more meaningful product if I phosphorylated the forward primer.otherwise it could be possible that the same primer could act as the reverse too, it being non-specific and degenerate and all....I never thought the forward primer I used could act as the reverse, but the sequence results speak otherwise!

[relaxin]

It is not uncommon to get a PCR product with one primer sequence at both ends. This product usually has a nonspecific sequence, which is not worth pursuing.

[mchlbrmn]

Each primer and DNA strand has a 5' to 3' orientation, that the polymerase "sees", and only extends the 3' end. You'll notice that your PCR product with the same primer at both ends had the 3' end primer in reverse complementary orientation (when reading one strand sequence) so that at both only the 3' end of that primer was extended. When the primer is "searching" for somewhere to anneal and extend, it can't tell the difference between one strand or the other, but just "looks" at all DNA for somewhere that it can find a sequence that it matches well enough at that temperature and salt concentration to anneal to. If the 3' end matches well enough, it will extend. If it finds perfect matches close enough to each other on the sense and and antisense strands, and if both are oriented so the 3' ends point toward each other, a PCR product will be generated (correction: both 3' ends MUST be oriented towards each other, since DNA must anneal to another strand in the opposite orientation). The primer does not realize that it's twin sister already primed the other strand. If the temperature is far enough below the Tm of the primer, a PCR product can be formed even if the primer has one, or several mismatches to the template. I found I needed to draw this out on paper, at first, to understand it.

[sindhu_kuttan]

Righto, so no PNK required for the primers I guess. Thanks a lot relaxin and mchlbrmn

[relaxin]

If you want to clone the PCR fragment into a plasmid vector, you need to treat the PCR fragment with PNK, because the vector will be dephosphorylated.