David H. Koch Center, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.
PLoS One. 2009 Dec 17;4(12):e8338. doi: 10.1371/journal.pone.0008338.
Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.
METHODOLOGY/PRINCIPAL FINDINGS: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6) ligand sequences.
CONCLUSIONS/SIGNIFICANCE: Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.
组合噬菌体展示技术在过去 20 年中被用于鉴定蛋白配体和蛋白-蛋白相互作用，揭示相关的分子识别事件。组合噬菌体展示文库选择的限速步骤是：（i）转导单位的计数，（ii）编码显示配体的测序。在这里，我们采用新兴的基因组技术来最小化这些挑战。
方法/主要发现：我们通过应用实时 PCR 进行快速定量来提高效率，从而实现无细菌噬菌体展示文库筛选，并添加噬菌体 DNA 下一代测序进行大规模配体分析，报告了一套完全集成的高通量定量和分析工具。该方法的劳动强度要小得多，并且允许进行严格的定量；对于医疗应用，包括在患者中的选择，它还代表了在文库给药后更长时间内从患者来源的活检或尸检中对数十万靶向颗粒进行定量分布分析和配体鉴定的一种进步。与当前方法相比，该方法的其他优点包括更高的灵敏度、更低的变异性、增强的线性、可扩展性和准确性，成本也大大降低。通过 qPhage 加焦磷酸测序获得的序列与通过传统 Sanger 测序的转导单位（TU）产生的数据集相似，不存在由于 GC 含量、密码子使用、氨基酸或肽频率引起的偏差。这些工具允许以 >1000 倍的速度更快地进行噬菌体展示选择和配体分析，并且生成 106 个配体序列的成本降低约 250 倍。
结论/意义：我们的分析表明，虽然这种方法与传统的菌落计数相关，但它也能够进行更大的采样，从而能够更快、更经济、更准确和一致地分析噬菌体的富集。总体而言，qPhage 加焦磷酸测序优于 TU 计数加 Sanger 测序，并且在体外、细胞内和体内的广泛噬菌体展示应用中被提议作为首选方法。
