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TIBS 1 5 - N O V E M B E R 1 9 9 0


Misuse of PCR errors due to its proof-reading activity.

Several independent clones must be
Its spectacular success over the last
30 years suggests that significant progress
sequenced in any case. If the initial DNA can be made even in the absence of an
Polymerase chain reaction (PCR) seems established theoretical foundation ...
material consists of only a few molecules
set to become a universal technique in Given the ease and simplicity of PCR
or if it is highly damaged (ancient amplification, it can be said to allow the
DNA analysis and manipulation. Invented
samples), these clones should practice of molecular biology without a
for tasks not manageable by other means, preferentially originate from different PCR permit.
PCR was promptly applied to make
reactions, since mutations introduced in
standard procedures easier and quicker. the first few cycles will be found in the Let us hope that he is wrong and that,
Now it is even used to facilitate trivial
great fraction of molecules at the end. eventually, intelligent application will
tasks such as whole genomic DNA
There are tasks which cannot be succede seductive, uncritical utilization
isolation and subcloning. However, the carried out in any other way than by PCR. of this powerful technique.
speed and versatility of the new But the benefits of PCR should be
technique has its price: the polymerase carefully weighed against its drawbacks References
makes errors, amplifies them and when its product is to be cloned. The use 1 Keohavong, P. and Thilly, W. G. (1989) Proc.
introduces new errors in each cycle (for of PCR to amplify genomic fragments for Natl Acad. Sci. USA 86, 9253-9257
Taq polymerase mostly A.T to G.C cloning in cases where sufficient amounts 2 Belyavsky,A., Vinogradova, T. and Rajewsky, K.
transitions), resulting inevitably in (1989) Nucleic Acids Res. 8, 2919-2932
of DNA can be easily isolated, to amplify
0.3--0.8% accumulated mutations after the 3 Meyerhans, A., Vartanian, J-P. and Wain-Hobson,
sequences from a standard cDNA library S. (1990) Nucleic Acids Res. 18, 1687-1691
usual 20-30 cycles 1,2. In addition to
(instead of amplifying and screening the 4 Shuldinger, A. R., Nirula, A. and Roth, J. (1989)
generating point mutations, PCR is known library) or even to facilitate subcloning(!) Nucleic Acids Res. 17, 4409
to promote formation of artificial 5 Fordham-Skelton,A. P., Yarwood, A. and Croy,
between recombinant phages and
recombinants by co-amplifying different plasmids 7 is hardly beneficial to anyone R. D. R. (1990) Mol. Gen. Genet. 221,
DNA fragments 3,4. The high error rate 134-138
else than to companies producing PCR 6 Keohavong, P., Kat, A. G., Cariello, N. F. and
does no harm when the whole product is kits and temperature cyclers.
analysed on a gel, used as a hybridization Thilly, W. G. (1988) DNA 7, 63-70
Davies and Pugsleys complain that we 7 Herrmann, J., Lee, P., Saya, H. and Nakajima,
probe or even sequenced. The danger are in danger of becoming a 'kit M. (1990) BioTechnictues 8, 376-380
begins with cloning of PCR products, by generation' of biologists who fail to 8 Davies, J. and Pugsley, A. (1990) Trends
which a randomly picked fragment from Biochem. Sci. 15, 137
understand the simplest principles of the
the mixture of mutants is conserved. Only 9 Erlich, H. A. (1990) in PCR Technology (Erlich,
techniques they use, since commercial H. A., ed.), p. 5, Stockton Press
a minority of researchers recognizes this
kits (instruction leaflet included)
critical point and keeps in mind the initial
guarantee success without thinking. Is the
PCR amplification through the whole 'life- PETR KARLOVSKY
misuse of PCR technology a manifestation
span' of the recombinant clone. It may
of this phenomenon? The editor of one of Abteilung for Molekulare Genetik der Gesellschaft
later be sequenced, perhaps in another
the PCR Bibles seems to admit it quite for Strahlen- und Umweltforschung,
laboratory, and if the mutagenic PCR frankly. He says about molecular biology9: Grisebachstrasse 6, D-3400 Goettingen, Germany.
treatment is not clearly indicated, the
sequence will be submitted to a database
without any warning note. The
consequences could be fatal.
Comparisons of alleles conferring
different phenotypes with their wild-type
counterpart, for instance, will be totally
misinterpreted. A polluted.sequence in a
database may mislead generations of
What should we do about this horror?
No one has to renounce PCR, but we
Errors in representing ways of distinguishing right- and left-
handed helical forms. As they point out,
should be aware that a clone obtained by protein and nucleic acid these diagrams are not necessarily
'wrong', but it is unfortunate that many
PCR is not a full-value clone equivalent to
classical cloning products, simply structures textbooks do not present these structures
as they appear in nature.
because amplification of DNA in vitro is
five to seven orders of magnitude less In his recent Textbook Error article,
accurate than replication of plasmids and Edison I implies that the representation of
phages in E. c o i l In agreement with ~-sheet structures drawn with D-amino References
1 Edison, A. S. (1990) Trends Biochem. Sci. 15,
Fordham-Skelton and co-workers 5, acid residues, rather than the natural
nucleotide sequence of a PCR clone L-amino acid residues, is an error that has 2 Day, R. A. and Ritter, E. J. (1967) J. Chem.
should be regarded only as a confirmation lain undiscovered for 29 years. This is not Educ. 44, 761-763
of its identity as a probe for screening so!
DNA libraries, not as an authentic gene Day and Ritter discussed this 'mistake'
sequence. If the PCR product is to be in a paper published in 19672. Their paper GERALDINE E. INDGE
cloned and the sequence is important, also gives examples of errors in the
Taq polymerase can be replaced by T4 representation of the sense of DNA and Stockport College of Technology, Stockport
DNA polymerase ~, which makes less protein helices, and describes simple SK1 3UQ, UK.