incredible PCR results, who can help explain?

D. KIM dkim at NMSU.Edu
Mon Dec 14 13:27:24 EST 1998

There was a pair of papers in recent Nucleic Acids Research in which was
described de novo DNA synthesis using Pfu polymerase plus nucleotides with
no template or primers.  They took the precaution of DNAase-treating the
reaction mix before cycling (to remove residual DNA in the enzyme prep).
Also works for Tth polymerase.  the time scale is long (over 2 hours,
maybe 4 hours before you see results).  DNA can be up to 20 kb long, and
consists of repeat units whose composition and unit length is dependent on
temperature and Mg/Mn concentration.

How about that?

Daniel Kim

supernova at wrote:
: Who has met the problem that a PCR reaction without any template can also
: generate some incredible products? Our product is a series of primer repeats.
: Our primer is a 30-mer designed by Primer Premier. And the both ends of the
: primer are “A”s. What ridiculous is that the product is a head-to-tail
: repeats. It deletes one base of “A” at the head/tail and then ligate the
: remaining, like the generation of a peptide bond–delete something and then
: ligate. Our PCR reaction is performed in a Clontech’s Polymerase Mix system.

: The PCR profile is:
: 94 Centigrade x 0.5min for denaturing;
: then touchdown at 72C and 70C respectively of 5 cycles, 2.5min for each cycle;
: finally at 68C for 30 cycles.

: I think the primers can’t form a dimer (if this kind could be regarded as a
: dimer) at the temperature around 70C. Furthermore, it’s not base
: complementation here, neither annealing. And only knockout ONE base each time.
: It’s incredible and unexplainable. The product is confirmed by sequencing.

: Who can help me then?

: -----------== Posted via Deja News, The Discussion Network ==----------
:       Search, Read, Discuss, or Start Your Own    

More information about the Methods mailing list