Characterization of the N+3 stutter product in the trinucleotide repeat locus DYS392

Stutter products generated during DNA amplification by the polymerase chain reaction (PCR) may complicate mixture interpretation. The PCR amplification of the DYS392 locus typically results in three distinct detectable PCR products: the true allele product (N), a stutter product three bases smaller (N-3), and a reproducible low-level product, three bases larger (N+3). Sequence analysis of the N+3 product demonstrated that its sequence is one TAT repeat longer than the true allele product. Our experiments demonstrated that the quantity of both N-3 and N+3 stutter increased as the allele number increased. The percent stutter also increased as the magnesium concentration was increased in the reaction, as well as when the amount of input DNA was decreased. As both stutter products behave in a similar and reproducible fashion, the same rules that apply to the interpretation of N-3 stutter products in short tandem repeat analysis, can be applied to N+3 stutters. The characterization of the DYS392 N+3 product is the first detailed published study of a stutter product larger than the true allele.