An Unexpected Product From Polymerase Chain Reaction-Mediated Site-Directed Mutagenesis Due to Misalignment of the...

Background: The I1307K (T3920 --> A) variant of the APC gene has been identified as a potential risk factor for colorectal cancer and is present in 6% of Ashkenazi Jews. Screening for this mutation may allow identification of people at elevated risk who would benefit from increased surveillance. Methods and Results: We designed an assay to detect the T3920 --> A allele using a primer mismatched at the 3 < 9 terminal nucleotide in the polymerase chain reaction (PCR) to generate a recognition site for the restriction enzyme Mse I. After optimization of the PCR for magnesium ion concentration and annealing temperature, the amplicon did not cut completely with the restriction enzyme in each of four tested DNAs. Sequence analysis of the PCR product that was resistant to digestion revealed that the T3920 --> A variant was not present. The artifact was caused by a single nucleotide loop-out in the genomic DNA template under the 3 < 9 region of the primer, which allowed the 3 < 9 terminal base of the primer to hybridize properly. As a result, the mismatched primer created a modified product different from that originally planned. At a magnesium ion concentration below the optimum for product yield, most of the product was digested by Mse I. Sequence analysis showed that, under these conditions, the intended product was produced. Conclusions: Mismatched primers can produce unintended products in a PCR due to looping out of a nucleotide in the template or the primer. The magnesium ion concentration can influence the sequence and amount of the product.