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Review
. 2011 Feb 15;16(2):1625-41.
doi: 10.3390/molecules16021625.

DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods

Thomas Lindner  1 , Harald Kolmar, Uwe Haberkorn, Walter Mier
Affiliations
Review

DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods

Thomas Lindner et al. Molecules. .

Abstract

Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

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Figures

Figure 1
Figure 1
Solid phase oligonucleotide synthesis cycle. DMT = 4,4’-dimethoxytrityl;B,B’ = protected nucleobase. Key: (i) activation (e.g., with tetrazole); (ii) iodine-oxidation; (iii) trichloroacetic acid deprotection; (iv) NH3-cleavage.
Scheme 1
Scheme 1
Preparation of trinucleotide building blocks. (i) MSNT-coupling; (ii) DMT-cleavage; (iii) phosphoramidite formation using a phosphordiamidite reagent.
Figure 2
Figure 2
Schematic presentation of the introduction of foreign DNA; (i) annealing and PCR; (ii) separate double digestion, followed by DNA-ligation with an appropriate vector in digested form.
Figure 3
Figure 3
Schematic illustration of the results obtained with clones possessing different propagation rates.
Figure 4
Figure 4
Enrichment of binding phages by affinity selection.
Figure 5
Figure 5
Schematic representation of the different types of phages with respect to their target binding and evolutionary fitness.
Figure 6
Figure 6
Schematic representation of the relative amplification of unspecific binders with a high propagation rate versus selective binders with moderate propagation rates after one panning round.
Figure 7
Figure 7
Schematic representation of the effect obtained by applying negative panning prior to the positive selection step.
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