A high-throughput phage display screening method using a combination of real-time PCR and affinity chromatography.
Phage display is one of the best methods to identify drug targets, although technical problems including imprecision in quantifying phage and false-positive results are common. To address these difficulties, we propose two methods to more rapidly identify drug-binding phage particles. First, quantification of phage using SYBR Green Real-time PCR significantly improved accuracy and reproducibility. Second, affinity-column chromatography for selection of drug-binding phage particles concentrated particles more than a 96-well plate, making a phage amplification step, which can bias phage distribution, unnecessary. The methods proposed here should be suitable for high-throughput phage-display screenings and ultimately lead to more rapid identification of drug targets.
Morohashi
Kengo
K
Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Arai
Tsuyoshi
T
Saito
Seiich
S
Watanabe
Madoka
M
Sakaguchi
Kengo
K
Sugawara
Fumio
F
D001435:Bacteriophages / Q000737:chemistry* / Q000187:drug effects; D053119:Benzophenanthridines; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D015394:Molecular Structure; D019151:Peptide Library; D010617:Phenanthridines / Q000737:chemistry / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D053119:Benzophenanthridines; D004269:DNA, Bacterial; C108284:NK 109; D019151:Peptide Library; D010617:Phenanthridines