Why are PCR products more difficult to clone into a DNA vector than an
insert that has simply been cut out of a construct?

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Clarification of Question by genemaster-ga on 03 Apr 2003 02:12 PST

Blunt ended ligation works reasonably well (you can screen using
directional PCR to overcome the orientation problem), but if you cut
the PCR product at the built in restriction enzyme sites, it still
doesn't go in as easily as it does using an insert cut from a plasmid,
especially if the PCR product is amplified from cDNA.  I was wondering
why this is so and how it can be overcome.

You can use blunt end ligation, but usually, if you've used a
restriction enzyme to cut the fragment out, it'll go straight into the
vector.  To do this with PCR you'd have to build the restriction site
into the primers.

There's also the issue of orientation.  With different restriction
sites at each end, you can define the orientation of the insert.  With
blunt end ligation, or the same type of site, you have to go fishing
among the transfectants for the one you want using RFLPs or
sequencing.

Hmmmm.
I wonder if it is a reagent carry over issue - something from the PCR
inhibiting the ligase.  Ahhh - how do you clean-up your PCR?  If you
just do an ethanol or isopropanol prep, you'll get left over primers
too.  If these contain the restriction site, they'll compete for the
ends of the vector.  You wouldn't have this issue going straight from
a restriction digest to a ligation.  You could try using resin spin
columns to recover the PCR product, or just cut it out of an agarose
gel and then clean it up.

Of course, if you are doing something similar already, I'll have to
have another think.

Some restriction enzymes require more DNA around the cut site than
just the consensus sequence - if the restriction site is on the far
end of the primers, perhaps the enzyme is not efficiently cutting the
fragments.

You might want to examine the quality of your nucleotides.  Just for
fun, you might want to create PCR products using 4 different companies
PCR-grade nucleotides to see if there are differences in cloning
success rates.  There are SIGNIFICANT differences in quality from
company to company that can affect both the PCR reaction and
downstream applications.  I also agree w/ Xarqi-ga in that the method
of "PCR Cleanup" will also greatly affect cloning.  Gel purified PCR
product is the standard...but once again, the quality of gel purified
product will vary greatly depending upon which companies kit you use. 
I obviously have my favorites but I do not want to make this a
commercial.

To answer your question, I do not agree that PCR amplified DNA will
necessarily be more difficult to clone than RE cut DNA.

Good luck w/ your cloning

For blunt ended cloning see Invitrogen and their TOPO cloning vectors.
Ligase independent and worked 6/7 times first time with my blunt ended
clones. Seventh one worked on second attempt. It's expensive but,
saves alot of time, effort and worry.

Thanks to all those who posted their comments and tips - this is just
a follow up to my question to let you know what I found the problem to
be.  Basically I was PCRing from a cDNA preperation and trying to
insert the allele into a vector.  Beacause I was getting a
primer/dimer problem (due to improperly designed primers) I was gel
purifying the PCR product, adding it to a double digest reaction, and
re-gel purifying the digested PCR product to get it away from the cut
off ends.

For those of you who haven't done that much cloning before you may not
know that agerose REALLY REALLY screws up ligation reactions, so
putting my insert through 2 rounds of gel purification (and not taking
enough effort to minimise the amount of agerose excised with the DNA)
prevented me from sticking it to the vector.

Ah well, you live and learn...