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Methods for Genetic Testing II
Published in Peter G. Shields, Cancer Risk Assessment, 2005
Laura Gunn, Luoping Zhang, Martyn T. Smith
One recently published assay used genotype selection for the detection of mutations in the H-RAS gene. By combining two previously published methods (8,9), the MutEx + allele-specific competitive blocker PCR (ACB-PCR) technique (10) is one of the most sensitive methods of genotypic selection. This assay begins with the denaturation of a heterogenous sample of mutant and wild-type double-stranded DNA. When reannealing, mutant DNA forms heteroduplex DNA with normal strands, while normal DNA strands form homoduplexes. Mut S, a thermostable protein, is added which binds to the mispaired sequence of the heteroduplex which protects the short sequence of mutant DNA from digestion from 3’-5’ exonuclease activity of T7 DNA polymerase, whereas the wild-type DNA is digested. This Mut-Ex step results in a 1000-fold enrichment of mutant alleles relative to wild type. To further increase sensitivity, the next step utilizes an additional selection technique, ACB-PCR. This genotypic selection method is based on preferential amplification by allele-specific primers. The first primer has more mismatches to wild type than mutant, resulting in preferential amplification of mutant DNA. The second primer is blocker primer, which preferentially anneals to the wild-type sequence, but is modified with a 3’-dideoxyguano-sine residue, which prevents extension. The ACB-PCR method therefore results in preferential amplification of mutant with a sensitivity of as few as 10 mutant alleles detected in the presence of 108 copies of the wild-type allele.
The Single-Stranded DNA Binding Protein of Bacteriophage T4
Published in James F. Kane, Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
Daniel H. Doherty, Peter Gauss, Larry Gold
We first note the effect of gp32 on a simple replication system that uses partially single-stranded lambda DNA (generated by exoIII digestion) as template and only one other protein of the T4 replication system, DNA polymerase.19 In this system (which mimics lagging-strand synthesis), gp32 stimulated DNA synthesis by the T4 DNA polymerase five to ten fold. Stimulation was greatest at low temperature and/or high salt concentration, conditions that favor intramolecular secondary structure in the template. The stimulation was interpreted as resulting from the removal of secondary structures present in the DNA template. However, denaturation of intramolecular structures is not sufficient to stimulate heterologous polymerases. The T4 single-stranded DNA binding protein does not stimulate E. coli DNA polymerase II but the E. coli single-stranded DNA binding protein (ssb gene product) does.58 This suggests that each DNA polymerase prefers single-stranded DNA with a conformation imposed by its homologous single-stranded DNA binding protein. The different contour lengths for DNA-protein bound complexes with gp32 and ssb (4.6A/nucleotide26 compared to 1.8A/nucleotide58) might reflect the base distortion required by the homologous polymerases for maximal stimulation of DNA synthesis in vitro (and perhaps in vivo). These results might also suggest that the stimulation of homologous polymerases is the result of direct protein-protein interactions between polymerase and single-stranded DNA binding protein. Such an interaction has been observed between gp32 and T4 DNA polymerase19,59 (and see below). However, we note that protein-protein complexes have not been observed betwen the T7 DNA polymerase and T7 DNA binding protein.60
Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies
Published in mAbs, 2021
Romain Rouet, Ohan Mazigi, Gregory J. Walker, David B. Langley, Meghna Sobti, Peter Schofield, Helen Lenthall, Jennifer Jackson, Stephanie Ubiparipovic, Jake Y. Henry, Arunasingam Abayasingam, Deborah Burnett, Anthony Kelleher, Robert Brink, Rowena A. Bull, Stuart Turville, Alastair G. Stewart, Christopher C. Goodnow, William D. Rawlinson, Daniel Christ
m396, CR3022, CR3014, and 80 R scFv were gene synthesized (Genscript) and cloned into the pHEN1 phagemid vector. Site-directed mutagenesis was carried out by Kunkel mutagenesis.19 In brief, phagemid vectors were transformed into E. coli CJ236, and a single colony grown in 2xYT media supplemented with 100 µg/mL ampicillin, 10 µg/mL chloramphenicol and 2% glucose until reaching an OD600nm of 0.4. Bacteria were then infected with KM13 helper phage and grown overnight at 30°C in 2xYT media supplemented with 100 µg/mL ampicillin, 10 µg/mL chloramphenicol, 50 µg/mL kanamycin and 0.25 µg/mL uridine. Phage particles were precipitated from the culture media using polyethylene glycol (PEG)/NaCl, and uridine-containing single-stranded DNA (dU-ssDNA) extracted using a QIAprep spin M13 kit (Qiagen). Mutagenesis was carried out by annealing degenerated oligonucleotides to the dU-ssDNA, followed by synthesis of the covalently closed circular DNA (cccDNA) with T7 DNA polymerase and T4 ligase (NEB). Finally, the cccDNA was transformed into electro-competent E. coli TG1 and bacteria titrated onto 2xYT agar plates containing 2% glucose and 100 µg/ml ampicillin to determine library sizes. Bacteria were harvested from the agar plates, grown in 2xYT media supplemented with 100 µg/mL ampicillin, and 2% glucose until reaching an OD600nm of 0.4. At this point, bacteria were infected with KM13 helper phage and grown overnight at 30°C in 2xYT media supplemented with 100 µg/mL ampicillin, and 50 µg/mL kanamycin. Phage antibody libraries were precipitated from culture media using PEG/NaCl and stored at 4°C.
Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models
Published in OncoImmunology, 2019
Daniel Newsted, Sunandan Banerjee, Kathleen Watt, Sarah Nersesian, Peter Truesdell, Levi L. Blazer, Lia Cardarelli, Jarrett J. Adams, Sachdev S. Sidhu, Andrew W. Craig
A mammalian expression construct was designed for expression of amino acids 23–534 of the human TGFBR2 ECD in frame with an IL-2 signal sequence at the N-terminus and human IgG1 Fc domain at the C-terminus using the pFuse2 vector (InvivoGen, San Diego, CA, USA). The construct was expressed and affinity purified from conditioned media of FreeStyle™ 293-F cells (InvivoGen) following transient transfection using XtremeGene HP (Roche). The mouse TGFBR2-Fc (catalog number 532-R2) and human TGFBR1-Fc (catalog number 3025-BR) were purchased from R&D Systems. The ECD-Fc fusion protein was used as the antigen to screen a phage displayed synthetic humanized Fab library.18 Binding selections, phage ELISAs and Fab protein purification were performed as described.41,42 Briefly, 1013 phage from a synthetic Fab library were cycled through rounds of binding selection with the antigen immobilized on 96-well Maxisorp Immunoplates (Fisher Scientific, Nepean, ON, Canada). After four rounds of positive selection, preceded by negative selection using human IgG1 Fc domain alone, phages from round 4 were produced as individual Fab-phage clones in 96-well format and Fab-phage ELISAs were performed to detect specific binding clones. Clones with positive binding to TGFBR2-Fc compared to Fc domain were subjected to DNA sequencing. The genes encoding for variable heavy- and light-chain domains of unique positive clones were sub-cloned into vectors designed for production of Fab or hIgG1 proteins in Escherichia coli or transfected FreeStyle™ 293-F cells, respectively, and proteins were affinity-purified on Protein A columns (GE Healthcare, Mississauga, ON, Canada). Affinity maturation phagemid libraries were created using optimized procedures that allow for the rapid construction of very large phage-displayed antibody repertoires (> 1010 unique clones).41,42 A dut−/ung−E. coli CJ236 host was transformed with template plasmid (5775) and helper phage. The result of transforming a dut−/ung− host was the propagation of phage encapsulating uracil-containing ssDNA (dU-ssDNA) which was then purified by precipitation with polyethelyne glycol. Oligonucleotides containing the mutagenic sequences for soft-randomizing CDRs of 5775, flanked by fifteen base pairs of sequence complementary to the annealing region, were annealed to the dU-ssDNA template of 5775. After annealing, the mutagenic oligonucleotides served to prime synthesis by T7 DNA polymerase of a DNA strand complementary to the uracil-containing template strand, forming a synthetic daughter strand lacking uracil. T4 DNA ligase ligated the synthesized DNA fragments, forming covalently closed circular double-stranded heteroduplex DNA (CCC-dsDNA). CCC-dsDNA was desalted and concentrated in preparation for electroporation. Selections using the affinity matured library were performed as earlier with increased stringency of washes and incorporating a final incubation of TGFBR2-Fc-bound phage with solution-phase TGFBR2-Fc (200 μg/mL) in round 4 to enrich for clones with slow off-rates. Estimation of affinity for selected matured phage clones was performed using high-throughput competitive phage ELISA.43