Partial strands synthesizing leads to inevitable aborting
and complicated products in consecutive polymerase
chain reactions (PCRs)
LUO Rui1,2 & ZHANG DaMing1†
1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093,
China;
2
Graduate University of Chinese Academy of Sciences，Beijing 100049, China
Various abnormal phenomena have been observed during PCR so far. The present study performed a
series of consecutive PCRs (including many rounds of re-amplification continuously) and found that
the abortion of re-amplification was inevitable as long as a variety of complicated product appeared.
The aborting stages varied, according to the lengths of targets. Longer targets reached the abortion
earlier than the shorter ones, marked by appearance of the complex that was immobile in
electrophoresis. Denatured gel-electrophoresis revealed that the complex was mainly made up of
shorter or partially synthesized strands, together with small amounts of full-length ones. Able to be
digested by S1 nuclease but unable by restriction endonucleases (REs), the complex was proved to
consist of both single regions and double-helix regions that kept the complex stable
thermodynamically. Simulations gave evidence that partial strands, even at lower concentration, could
disturb re-amplification effec- tively and lead to the abortion of re-amplifications finally. It was pointed
out that the partial strands formed chiefly via polymerase’s infidelity, and hence the solution to lighten
the abnormality was also proposed.
consecutive PCR, abnormal complicated product, partial strand synthesis, disturbing effect, PCR-mediated recombination, long distance PCR
PCR and PCR-derived methods have a wide range of
applications, such as target-sequence amplification,
cDNA-library construction, directed-mutagenesis, probe
labeling, genetic analysis, clinical diagnoses, and
forensic detection[1]. PCRs have become basic
techniques in molecular biology. However, lots of
abnormal phenomena have been observed in the PCR
process or for amplified products, such as nucleotide
substitution[2], bias[3], recombination[4,5], slippage[6], and
jumping[7]. And similarly, various difficulties were
encountered and some special treatments were needed
when the amplifying target was very long
(Long-Distance PCR)[8,9].
In most cases, only one round of PCR was enough to
obtain enough expected specific products. But re-amplification in further rounds was necessary in certain
cases. For instance, the amount of the expected product
1
Erlich H A, Gelfand D, Sninsky J J. Recent advances in the
was too little, or products contained various non-specific
sequences in the first round of PCR. The reamplifying of
desired bands was also necessary in AFLP or DD-PCR.
One kind of results that was often obtained by re-amplifications was the appearance of a range of smears in
electrophoresis. Various treatments had been applied to
—
resolving or alleviating this abnormality[10 12]. Bell &
DeMarini analyzed this phenomenon and concluded that
it was the annealing of the 3′-OH ends of the product
that served as “primers” to genomic templates or to the
products themselves that produced longer randomlengths of fragments during the later circles of PCR[13].
Received May 1, 2001; accepted June 01, 2001
doi: 10.1007/s11427-007-0043-z
†
Corresponding author (email: zhangdm@ibcas.ac.cn)
Supported by the National Natural Science Foundation of China (Grant No.
30430030)
polymerase chain reaction. Science, 1991, 252: 1643―1651
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Keohavong P, Thilly W G. Fidelity of DNA polymerases in DNA
amplification. Proc Natl Acad Sci USA, 1989, 86: 9253―9257
Mutter G L, Boynton K A. PCR bias in amplification of androgen
receptor alleles, A trinucleotide repeat marker used in clonality
studies. Nucleic Acids Res, 1995, 23: 1411―1418
Jansen R, Ledley F D. Disruption of phase during PCR amplification
and cloning of heterozygous target sequences. Nucleic Acids Res,
1990, 18: 5153―5156
Meyerhans A, Vartanian J P, Wain-Hobson S. DNA recombination
during PCR. Nucleic Acids Res, 1990, 18: 1687―1691
Kunkel T A. Frameshift mutagenesis by eucaryotic DNA polymerases
in vitro. J Biol Chem, 1986, 261: 13581―13587
Paabo S, Irwin D M, Wilson A C. DNA damage promotes jumping
between templates during enzymatic amplification. J Biol Chem,
1990, 265: 4718―47
Cheng S, Fockler C, Barnes W M, et al. Effective amplification of
long targets from cloned inserts and human genomic DNA. Proc Natl
Acad Sci USA, 1994, 91: 5695―5699
Thiel V, Rashtchian A, Herold J, et al. Effective amplification of
20-kb DNA by reverse transcription PCR. Anal Biochem, 1997, 252:
62―70
Hengen P N. Reamplification of PCR fragments. Trends Biochem Sci,
1995, 20: 124―125
Bonnet S, Prevot G, Bourgouin C. Efficient reamplification of
differential display products by transient ligation and thermal
asymmetric PCR. Nucleic Acids Res, 1998, 26: 1130―1131
Frost M R, Guggenheim J A. Prevention of depurination during
elution facilitates the reamplification of DNA from differential
display gels. Nucleic Acids Res, 1999, 27: 1386―1391
Bell D A, DeMarini D M. Excessive cycling converts PCR products to
random-length higher molecular weight fragments. Nucleic Acids Res,
1991, 19: 5079
Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual.
2nd ed. New York: Cold Spring Harbor Laboratory Press, 1995
Olsen D B, Eckstein F. Incomplete primer extension during in vitro
DNA amplification catalyzed by Taq polymerase; exploitation for
DNA sequencing. Nucleic Acids Res, 1989, 17: 9613―9620
Marton A, Delbecchi L, Bourgaux P. DNA nicking favors PCR
recombination. Nucleic Acids Res, 1991, 19: 2423―2426
Huang M M, Arnheim N, Goodman M F. Extension of base mispairs
by Taq DNA polymerase: Implications for single nucleotide
discrimination in PCR. Nucleic Acids Res, 1992, 20: 4567―4573
Bambara R A, Fay P J, Mallaber L M. Methods of analyzing
processivity. Methods Enzymol, 1995, 262: 270―280
Odelberg S J, Weiss R B, Hata A, et al. Template-switching during
20
21
22
23
24
25
26
27
28
29
30
31
32
33
DNA synthesis by Thermus aquaticus DNA polymerase I. Nucleic
Acids Res, 1995, 23: 2049―2057
Wang G C, Wang Y. Frequency of formation of chimeric molecules as
a consequence of PCR coamplification of 16S rRNA genes from
mixed bacterial genomes. Appl Environ Microbiol, 1997, 63:
4645―4650
Bradley R D, Hillis D M. Recombinant DNA sequences generated by
PCR amplification. Mol Biol Evol, 1997, 14: 592―593
Judo M S, Wedel A B, Wilson C. Stimulation and suppression of PCRmediated recombination. Nucleic Acids Res, 1998, 26: 1819―1825
Zaphiropoulos P G. Non-homologous recombination mediated by
Thermus aquaticus DNA polymerase I. Evidence supporting a copy
choice mechanism. Nucleic Acids Res, 1998, 26: 2843―2848
Cronn R, Cedroni M, Haselkorn T, et al. PCR-mediated
recombination in amplification products derived from polyploid
cotton. Theor Appl Genet, 2002, 104: 482―489
Hamilton S C, Farchaus J W, Davis M C. DNA polymerases as
engines for biotechnology. Biotechniques, 2001, 31: 370―376,
378―380, 382―373
Kelman Z, Hurwitz J, O'Donnell M. Processivity of DNA
polymerases: two mechanisms, one goal. Structure, 1998, 6:
121―125
Kornberg A. Ten commandments: lessons from the enzymology of
DNA replication. J Bacteriol, 2000, 182: 3613―3618
Fromenty B, Demeilliers C, Mansouri A, et al Escherichia coli
exonuclease III enhances long PCR amplification of damaged DNA
templates. Nucleic Acids Res, 2000, 28: e50
Bullard J M, Williams J C, Acker W K, et al. DNA polymerase III
holoenzyme from Thermus thermophilus identification, expression,
purification of components, and use to reconstitute a processive
replicase. J Biol Chem, 2002, 277: 13401―13408
Davidson J F, Fox R, Harris D D, et al. Insertion of the T3 DNA
polymerase thioredoxin binding domain enhances the processivity
and fidelity of Taq DNA polymerase. Nucleic Acids Res, 2003, 31:
4702―4709
Wang Y, Prosen D E, Mei L, et al. A novel strategy to engineer DNA
polymerases for enhanced processivity and improved performance in
vitro. Nucleic Acids Res, 2004, 32: 1197―1207
Kainz P, Schmiedlechner A, Strack H B. Specificity-enhanced
hot-start PCR: addition of double-stranded DNA fragments adapted to
the annealing temperature. Biotechniques, 2000, 28: 278―282
Lin Y, Jayasena S D. Inhibition of multiple thermostable DNA
polymerases by a heterodimeric aptamer. J Mol Biol, 1997, 271:
100―111
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