Nucleic Acids Research, Vol 26, Issue 12 2843-2848, Copyright © 1998 by Oxford University Press
PG Zaphiropoulos
RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite
orientations of exon 6, resulted in PCR products containing segments of
exons joined at non-consensus splice sites. Moreover, many of the PCR
products identified were composed of not only a single region containing
exonic segments joined at non-consensus splice sites but, instead, of
several repeats of the non-canonically joined region. To investigate
whether these PCR products represent pre-existing molecules or are
generated during the amplification process, the liver cDNA template was
replaced by a plasmid containing the P450 2C6 cDNA. Surprisingly, PCR
products containing repeats of non-canonically joined exonic segments were
again revealed. In some cases the position of this non-canonical joining
was a sequence of one or two identical nucleotides; however, there were
also a number of products lacking any nucleotide identity at the position
of joining. DNA nicking and/or DNA damage is thought to favour
recombination during PCR, probably by misalignment of incomplete DNA
strands; however, the presence of multiple repeats of the recombined region
in the PCR products identified suggests a certain repetitiveness of the
underlying mechanism. It is therefore proposed that these products result
from a template switching event that occurs several times during a single
polymerization step, following a rolling circle model of DNA synthesis.
ARTICLES
Non-homologous recombination mediated by Thermus aquaticus DNA polymerase I. Evidence supporting a copy choice mechanism
Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum, 141 57 Huddinge, Sweden. peza@cnt.ki.se
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