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. 2002 May 1;30(9):2083-8.
doi: 10.1093/nar/30.9.2083.

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'

Janelle R Thompson  1 , Luisa A Marcelino, Martin F Polz
Affiliations

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'

Janelle R Thompson et al. Nucleic Acids Res. .

Abstract

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.

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Figures

Figure 1
Figure 1
CDCE separation of heterologous PCR products (A) combined after amplification and (B–E) co-amplified. Peak 1, V.vulnificus; peak 2, V.parahaemolyticus; peak 3, V.cholera. Heteroduplex peaks are denoted by bracketed numbers corresponding to the identities of the single-stranded heterologs. The indicated primer is a 20mer with a 54mer GC-rich clamp. Relative fluorescent intensities are indicated on the y-axis. (A) Peak identification standard, three single-template PCR products combined after amplification. (B–D) Co-amplification products of two-species template mixtures. (E) Three-species template mixture, heteroduplexes (2,3) and (1,3) co-migrate.
Figure 2
Figure 2
Classes and frequencies of mosaic sequences possible from mismatch repair of heteroduplex containing three mismatches. (A) Mismatch repair of unmethylated heteroduplexes containing three mismatches can generate three classes of mosaic sequences: (i) restoration of an original sequence, (ii) creation of mosaics with distinct domains at the 3′ and 5′ ends and (iii) creation of mosaics with related insert termini but internal hybridism. (B) Relative frequencies of the mosaic classes produced by MutHLS repair of one heteroduplex containing three mismatches are indicated for independent repair of each mismatch or for co-repair of adjacent mismatches where repair initiates only at nick sites flanking the heteroduplex insert.
Figure 3
Figure 3
Mean number of classes observed in n samples. Curves calculated by iteration of the formula described by Ewens (18) where C(n) is the average number of sequence variants observed at sample n, Ctot is the total number of sequence variants in the population after cloning and mismatch repair, and p(i) is the probability of observing the ith sequence variant in the population. When 50, 5 and 0.1% of cloned inserts are heteroduplexes, mosaic sequences are detected at decreasing frequencies (A) via independent repair of adjacent mismatches by the MutHLS system, and (B) allowing co-repair of adjacent mismatches.
Figure 4
Figure 4
CDCE separation of three-template PCR amplification products before and after three-cycle reconditioning PCR. Peak identities are as indicated in Figure 1. (A) The peak areas of homoduplex and heteroduplex DNA indicate they are at similar proportions in the final PCR product (∼50% heteroduplexes). (B) Heteroduplex peaks are eliminated by a three-cycle reconditioning PCR such that their relative frequency is below the background detection limit (<1%), illustrating at least a 50-fold reduction in heteroduplex frequency.
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