Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner

Abstract

Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.

Figure 1

Sequencing analysis of PCR amplified genomic DNA of a patient with gastric cancer. DNA was isolated from morphologically normal and cancer cells of paraffin-embedded tissues. The nucleotides surrounding the reported G to A transition mutation in exon III of the UNG gene are shown. A: Wild-type sequence. B: Position of the G to A transition mutation shown by an arrow. We prepared serial dilutions of P1 phage and performed 10 independent PCRs for each titer. The approximate number of P1 vector used in each PCR mix was 50 copies (C), 25 copies (D). None of the 10 PCR products from the 50-copy titer showed the base substitution. D: Represents 6 of 10 PCR reactions (60%).

Figure 2

A–C: A region of the GC-rich exon IA that displayed high incidence of PCR-produced base substitutions at low DNA input. A: Wild-type sequence. In B and C, approximately 25 copies of the P1 phage were used as template in the PCR mixture. D–F: A GC-rich region in intron IA that showed high rate of PCR-induced G to A base substitution at very low DNA template concentration. D: Wild-type DNA sequence. In E and F, each PCR mix contained approximately 25 copies of P1 phage DNA template. The arrows show the site of base changes.