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A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.

ABSTRACT: We have determined diversities exceeding 10(12) different sequences in an annealing and melting assay using synthetic randomized oligonucleotides as a standard. For such high diversities, the annealing kinetics differ from those observed for low diversities, favouring the remelting curve after annealing as the best indicator of complexity. Direct comparisons of nucleic acid pools obtained from an aptamer selection demonstrate that even highly complex populations can be evaluated by using DiStRO, without the need of complicated calculations.

SUBMITTER: Schutze T

PROVIDER: S-EPMC2831324 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.

Schütze Tatjana T Arndt Peter F PF Menger Marcus M Wochner Aniela A Vingron Martin M Erdmann Volker A VA Lehrach Hans H Kaps Christian C Glökler Jörn J

Nucleic acids research 20091203 4

We have determined diversities exceeding 10(12) different sequences in an annealing and melting assay using synthetic randomized oligonucleotides as a standard. For such high diversities, the annealing kinetics differ from those observed for low diversities, favouring the remelting curve after annealing as the best indicator of complexity. Direct comparisons of nucleic acid pools obtained from an aptamer selection demonstrate that even highly complex populations can be evaluated by using DiStRO, ...[more]

PMID: 19965765

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Project description:A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 10(9) sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an alpha-synuclein ep-PCR library and NNS synthetic oligonucleotide libraries are given as examples.

Dataset Information

A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.

Publications

A calibrated diversity assay for nucleic acid libraries using DiStRO--a Diversity Standard of Random Oligonucleotides.

Similar Datasets

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