A new randomization assay reveals unexpected elements of sequence bias in model 'randomized' gene libraries: implications for biopanning.
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.
Palfrey
D
D
Pharmaceutical Sciences Research Institute, Aston University, Aston Triangle, Birmingham, UK.
Picardo
M
M
Hine
A V
AV
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D015723:Gene Library; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D009841:Oligonucleotides; D013942:Thymine Nucleotides; C037773:2',3'-dideoxyadenosine triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate