Cycled Primer Extension: A Method for DNA Amplification and Labeling from Templates of Unknown Sequence

Abstract

A method has been developed for simultaneous radio-labeling and amplification of DNA hybridization probes. The method is termed cycled primer extension (CPE). CPE is a series of temperature-driven reactions in which template DNA is successively denatured and extended by a thermostable primer-dependent DNA polymerase. The primers consist of semirandom nanomers of the form 5′-NNN NNN (G/C)(G/C)(G/C)-3′. These nanomers have the capacity to anneal to any template DNA and serve as initial anchors for extension at the high temperatures required for Taq DNA polymerase activity. CPE cycles consist of 94°C denaturation, annealing of primers to template upon ramping to 24°C, and gradual extension of the primer along the template as temperature is ramped back to 94°C. Labeling efficiency with [32P]dCTP was examined and optimized as determined by the relation to ratios of radiolabeled to unlabeled dCTP, by number of cycles, and by primer composition and sequence. CPE probes can be generated without regard to size or sequence of template and have a high specific activity (∼ 10 dpm/μg). With CPE, hybridization signals equivalent to those from random primed probes are routinely obtained with initial template amounts as low as 1 ng.