The invention provides methods and compositions for detecting and/or quantifying vector backbone in a nucleic acid preparation comprising a polynucleotide of interest using amplification assays that amplify a junction located between the polynucleotide of interest and the vector backbone, under conditions whereby amplification can occur, wherein the junction comprises a recognition site for a nuclease, and detecting the absence of an amplification product, whereby the absence of the amplification product indicates low or no vector backbone and/or quantifying the amount of amplification product to determine the amount of vector backbone in the nucleic acid preparation.