A new randomization assay reveals Unexpected elements of sequence bias in model 'randomized' gene libraries: Implications for biopanning

David Palfrey, M. Picardo, Anna V. Hine*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Although techniques such as biopanning rely heavily upon the screening of randomized gene libraries, there is surprisingly little information available on the construction of those libraries. In general, it is based on the cloning of 'randomized' synthetic oligonucleotides, in which given position(s) contain an equal mixture of all four bases. Yet, many supposedly 'randomized' libraries contain significant elements of bias and/or omission. Here, we report the development and validation of a new, PCR-based assay that enables rapid examination of library composition both prior to and after cloning. By using our assay to analyse model libraries, we demonstrate that the cloning of a given distribution of sequences does not necessarily result in a similarly composed library of clones. Thus, while bias in randomized synthetic oligonucleotide mixtures can be virtually eliminated by using unequal ratios of the four phosphoramidites, the use of such mixtures does not ensure retrieval of a truly randomized library. We propose that in the absence of a technique to control cloning frequencies, the ability to analyse the composition of libraries after cloning will enhance significantly the quality of information derived from those libraries. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Original languageEnglish
Pages (from-to)91-99
Number of pages9
JournalGene
Volume251
Issue number1
DOIs
Publication statusPublished - 13 Jun 2000

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Keywords

  • coupling efficiency
  • library
  • phage display
  • phosphoramidite mixtures
  • random

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