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PMID: 12219078 Published · ppublish English Comparative Study Evaluation Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Nucleic acid evolution and minimization by nonhomologous random recombination.

Nature biotechnology ·Vol. 20 ·No. 10 ·2002-10-00 ·Pages 1024-9

Bittker JA, Le BV, Liu DR

Abstract

We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure-activity relationships among evolved sequences.

MeSH Terms
Base Sequence Cloning, Molecular DNA/genetics DNA Primers Directed Molecular Evolution/methods Evolution, Molecular Molecular Sequence Data Nucleic Acid Amplification Techniques/methods Polymerase Chain Reaction Random Amplified Polymorphic DNA Technique/methods Recombination, Genetic Reproducibility of Results Sensitivity and Specificity Sequence Analysis, DNA/methods Sequence Homology, Nucleic Acid Streptavidin/genetics
Chemicals
DNA Primers DNA Streptavidin
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Bittker Joshua A
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Le Brian V
Liu David R
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Article Info
Journal
Nature biotechnology
Abbr.
Nat Biotechnol
ISSN
1087-0156
DOI
10.1038/nbt736
Published
2002-10-00
Epub
2002-00-09
Pages
1024-9
Language
English
Region
United States
NLM ID
9604648
PMCID
PMC2819268
Subset
IM
Grants
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1038/nbt736 PMC Full Text View journal details
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