期刊
BIOTECHNIQUES
卷 44, 期 3, 页码 351-+出版社
FUTURE SCI LTD
DOI: 10.2144/000112689
关键词
-
资金
- National Institutes of Healths National Cancer Institute [CA118591]
Standard systematic evolution of ligands by exponential enrichment (SELEX) protocols require libraries that contain two primers, one on each side of a central random domain, which allow amplification target-bound sequences via PCR or RT-PCR. However these primer sequences cause nonspecific binding by their nature (generally adding about 20 tit on each end of the random sequence of about 30-40 tit), and can result in large numbers of false positive binding sequences and/or interfere with good binding random sequences. Here, we have developed two DNA-based methods that reduce and/or eliminate the primer sequences from the target-binding step, thus reducing or eliminating the interference caused by the primer sequences. In these methods, the starting selection libraries contain a central random sequence that is: (i) flanked by only 2 nt on each side (minimal primer); or (ii) flanked only by either a 2- or 0-nt overhand on the 3'end (primer-free). These methods allow primer regeneration and re-elimination after and before selection, are fast and simple, and don't require any chemical modifications for selection in a variety of conditions. Further, the selection rounds are performed with DNA oligomers, which are generally employed as end product aptamers.
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