Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.
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.
Thompson
Janelle R
JR
Massachusetts Institute of Technology, Department of Civil and Environmental Engineering, Cambridge, MA 02139, USA.
Marcelino
Luisa A
LA
Polz
Martin F
MF
D016477:Artifacts; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D004706:Endodeoxyribonucleases / Q000737:chemistry; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis* / Q000096:biosynthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014733:Vibrio / Q000235:genetics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D012336:RNA, Ribosomal, 16S; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D045643:DNA Repair Enzymes