Elimination of Carryover Contamination during Nucleic Acid Amplification Based on Damaged Base Excision by DNA Repair...

Nucleic acid amplification is a pivotal technology for gene investigation in molecular biology as well as an indispensable tool for nucleic acid-based analyses across various disciplines. However, the aerosolized amplicons render the subsequent reaction susceptible to carryover contamination, leading to serious false positive results, particularly in scenarios involving repeated amplification of identical sequences. Although the uracil-DNA glycosylase-based strategy can address this issue for natural DNA amplification, the uracil-containing DNA required for bisulfite conversion-based DNA methylation analysis fails to be analyzed because both aerosol and the intended template can be hydrolyzed. In this study, an innovative strategy for one-pot elimination of carryover contamination is established, applicable to both natural and uracil-containing DNA analyses using either thermal cycle-based or isotherm-based nucleic acid amplification. By incorporation of deoxyinosine triphosphate, hypoxanthine is inserted into amplicons, resulting in a different base composition from the original template. Hypoxanthine, a damaged base as the recognition site of endonuclease V, acts as a label for aerosol cleavage, while the intended template lacking hypoxanthine is unaffected. By systematic optimization, abundant aerosols are thoroughly hydrolyzed within a brief time. Despite the use of deoxyinosine triphosphate as an aberrant deoxyribonucleoside triphosphate, the amplification efficiency, sensitivity, and specificity are insusceptible. In addition, the hypoxanthine-containing amplicons can be analyzed not only for length identification by gel and capillary electrophoresis but also for sequence identification by sanger sequencing, pyrosequencing, and massively parallel sequencing. Moreover, the hypoxanthine-containing amplicons can be further used for cloning and restriction enzyme digestion, indicating the significant potential of this anti-aerosol strategy.