AN UNEXPECTED PRODUCT FROM POLYMERASE CHAIN REACTION-MEDIATED SITE-DIRECTED MUTAGENESIS DUE TO MISALIGNMENT OF THE MISMATCHED PRIMER

Background: The I1307K (T3920-->A) variant of the APC gene has been id entified as a potential risk factor for colorectal cancer and is prese nt in 6% of Ashkenazi Jews. Screening for this mutation may allow iden tification 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' terminal nucleot ide in the polymerase chain reaction (PCR) to generate a recognition s ite for the restriction enzyme Mse I. After optimization of the PCR fo r magnesium ion concentration and annealing temperature, the amplicon did not cut completely with the restriction enzyme in each of four tes ted DNAs. Sequence analysis of the PCR product that was resistant to d igestion revealed that the T3920-->A variant was not present. The arti fact was caused by a single nucleotide loop-out in the genomic DNA tem plate under the 3' region of the primer, which allowed the 3' terminal base of the primer to hybridize properly. As a result, the mismatched primer created a modified product different from that originally plan ned. At a magnesium ion concentration below the optimum for product yi eld, most of the product was digested by Mse I. Sequence analysis show ed that, under these conditions, the intended product was produced. Co nclusions: 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.