Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries

Weihua Pan, Ping Xin, Gary Clawson

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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 of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), 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.

Original languageEnglish (US)
Pages (from-to)351-360
Number of pages10
JournalBioTechniques
Volume44
Issue number3
DOIs
StatePublished - Mar 1 2008

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SELEX Aptamer Technique
Gene Library
Ligands
Libraries
DNA
Chemical modification
Oligomers
Amplification
Polymerase Chain Reaction
Regeneration

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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abstract = "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 of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), 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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Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries. / Pan, Weihua; Xin, Ping; Clawson, Gary.

In: BioTechniques, Vol. 44, No. 3, 01.03.2008, p. 351-360.

Research output: Contribution to journalArticle

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