DEGRADABLE DUMP OUTER PRIMERS IN MERGED TANDEM (M T)-NESTED PCR - LOW-COPY AND SINGLE-COPY DNA TARGET AMPLIFICATION/

PCR amplification of DNA from a single initiating genomic molecule or low-copy template often requires two sequential amplification reaction s with nested primer pairs to achieve the necessary specificity and se nsitivity. Residual outer primers can result in undesired primer activ ity during the inner nested cycles. To circumvent this problem, me hav e used dU-containing primers for first round amplification and then ur acil N-glycosylase (UNG) to degrade them and the ends of their dU-prim er-containing amplified DNA products. We have applied this method to t he detection of an exon 11 mutation in the HEXA gene. We have merged t he step of a single-tube PCR amplification with outer dU primers with a tandem amplification using non-dU-nested primers (hence, the term me rged tandem-nested or M/T-nested PCR). Serial dilutions of genomic DNA showed that this method could amplify a specific target from as few a s three haploid genome equivalents of template DNA. Specific products were obtained from the DNA of single cells in 19 of 20 replicates, usi ng 12 outer and 28 inner nested PCR cycles, with an intervening UNG di gestion step, When coupled with heteroduplex mutational analysis, this method reliably distinguished mutant versus wildtype HEXA gene fragme nts amplified from single cells without primer artifact. (C) 1998 Acad emic Press.