Pyrosequencing® Protocols

Volume 373 of the series Methods in Molecular Biology™ pp 135-146

Pyrosequencing® of Phage Display Libraries for the Identification of Cell-Specific Targeting Ligands

  • Ahad A. RahimAffiliated withGene Therapy Group, Chester Beatty Laboratories, Institute of Cancer Research

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High combinatorial phage display libraries have become an important tool in the search for ligand-receptor interactions. The advantage this approach offers is the ability to screen large repertoires of peptides, displayed on the coat proteins of bacteriophages, against a target at the same time. In addition, no prior knowledge is required of the target or the ligand. However, to characterize the peptides of interest a short length of the bacteriophage genome that encodes the peptide sequence requires DNA sequencing. The number of candidate bacteriophages can be large and so sequencing is expensive, timeconsuming, and laborious. Therefore, a methodology using Pyrosequencing® has been developed where 96-phage displaying a seven amino acid peptide can be analyzed simultaneously within 45 min and at a fraction of the cost associated with traditional automated Sanger sequencing.