Cycled Primer Extension: A Method for DNA Amplification and Labeling from Templates of Unknown Sequence
✍ Scribed by G. Ruano; M.E. Lewis; R.E. Kouri
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 677 KB
- Volume
- 212
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
A method has been developed for simultaneous radiolabeling 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^{\circ} \mathrm{C}) denaturation, annealing of primers to template upon ramping to (24^{\circ} \mathrm{C}), and gradual extension of the primer along the template as temperature is ramped back to (94^{\circ} \mathrm{C}). Labeling efficiency with (\left[{ }^{32} \mathrm{P}\right]) 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 (\left(\sim 10^{9} \mathrm{dpm} / \mu \mathrm{g}\right)). With CPE, hybridization signals equivalent to those from random primed probes are routinely obtained with initial template amounts as low as (1 \mathrm{ng}). C 1983 Academic Press, Inc.