Counting alleles reveals a connection between ...

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TECHNICAL REPORTS Phage quantification by ELISA. Dilution series of phage were coated in 100 mM NaHCO3, pH 8.6 in MaxiSorb ELISA plates (Life Technologies, Karsruhe, Germany) for 16 h at 4°C. Blocking was done with 2% skim milk powder in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, pH 7.3) for 2 h at room temperature. Phage were detected with the monoclonal mouse antibody B62-FE2 (Progen, Heidelberg, Germany) specifically binding an epitope on the pVIII major coat protein of the M13 phage. M13KO7 phage of known colony-forming units were used for standardization. Immunoblot. Approximately 1010 phage were applied per lane on a 10% polyacrylamide gel. Blocking was done with 2% skim milk powder in PBS for 2 h at room temperature. Immunostaining was done with the mouse mAb anti-g3p (MoBiTec, Göttingen, Germany) recognizing the pIII coat protein of M13KO7, visualized with 3,3´, 5,5´-tetramethylbenzidene (TMB) substrate (Promega, Madison, WI). Antigen-binding phage ELISA. Dilution series of BSA-phOx antigen in 100 mM NaHCO3, pH 8.6, were coated in MaxiSorb ELISA plates (Life Technologies) for 16 h at 4°C. After blocking of unspecific binding with 2% skim milk powder in PBS for 2 h at room temperature, 2 × 109 single-chain phage diluted in 2% skim milk powder in PBS were applied to each well for 1 h at room temperature. After washing six times with 400 µl PBS per well, the bound phage were detected with mAb B62-FE2 as described above. Panning. A human scFv fragment library in pSEX81 with a calculated maximal complexity of 2 × 107 was generated from peripheral lymphocyte preparations as described21. Tetanus toxin obtained from Virotech (Rüsselsheim, Germany) was coated to six ELISA wells (Life Technologies) at a concentration of 0.1 µg/ml in 100 mM NaHCO3, pH 8.6, for 16 h at 4°C. The wells were blocked with 2% skim milk in PBS for 2 h at room temperature, after which 1011 phage of the library were applied to each well and incubated at 4°C overnight. After five washes with PBS/0.1% Tween and five with PBS, bound phage were eluted with 1 µg/ml trypsin (Life Technologies) in PBS for 15 min at room temperature. Eluted phage were used for the infection of 20 ml of E. coli XL1blue at a OD600 of 0.4. Infected bacteria were grown on Luria–Bertani agar plates containing glucose and ampicillin, scratched from the plates, and used for the production of antibody phage for the next round of panning as previously described21.

Acknowledgments The financial support from the Heidelberger Akademie der Wissenschaften (Germany) is gratefully acknowledged. We wish to thank M. Little for support, M. Bechtel from Genzyme Virotech GmbH for supplying the tetanus toxin, and Olaf Broders for helping with the experimental work. We are grateful for E.K.F. Bautz for his continuous support and suggestions. 1. Hoogenboom, H.R. et al. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19, 4133–4137 (1991). 2. Breitling, F. et al. A surface expression vector antibody screening. Gene 104, 147–153 (1991). 3. Marks, J.D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991). 4. Barbas, C.F. et al. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982 (1991). 5. Gavilondo, J.V. & Larrick, J.W. Antibody engineering at the millennium. BioTechniques 29, 128–145 (2000). 6. Fisch, I. et al. A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage. Proc. Natl. Acad. Sci. USA 93, 7761–7766 (1996). 7. Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000). 8. Sblattero, D. & Bradburry, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80 (2000). 9. Griffiths, A.D. et al. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12, 725–734 (1993) 10. Duenas, M. & Borrebaeck, C.A. Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries. FEMS Microbiol Lett. 125, 317–321 (1995) 11. Rakonjac, J., Jovanovic, G. & Model, P. Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3. Gene 198, 99–103 (1997) 12. Nelson, F.K., Friedman, S.M. & Smith, G.P. Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III. Virology 108, 338–350 (1981) 13. Crissman, J.W. & Smith, G.P. Gene-III protein of filamentous phages: evidence for a carboxyl-terminal domain with a role in morphogenesis. Virology 132, 445–455 (1984) 14. Dotto, G.P. & Zinder, N.D. Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein. Nature 311, 279–280 (1984).

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15. Davis, N.G., Boeke, J.D. & Model, P. Fine structure of a membrane anchor domain. J. Mol. Biol. 181, 111–121 (1985). 16. Rondot, S. Präsentation von Peptidepitopen auf rekombinanten F-Pili für eine antigenspezifische Infektion von Bakterien (Inaugural-Dissertation). (University of Heidelberg, Germany; 1997). 17. Dübel, S. et al. A family of vectors for surface display and production of antibodies. Gene 128, 97–101 (1993). 18. Diederich, L., Rasmussen, L.J. & Messer, W. New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome. Plasmid 28, 14–24 (1992) 19. Welschof, M. et al. The antigen-binding domain of a human IgG-anti-F(ab′)2 autoantibody. Proc. Natl. Acad. Sci. USA 94, 1902–1907 (1997). 20. Micheel, B. et al. Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages. J. Immunol. Methods 171, 103–109 (1994). 21. Koch, J. & Dübel., S. Generation of antibody libraries from human donors. In Antibody engineering, (eds Kontermann, R. & Dübel, S.) (Springer Verlag, Heidelberg/New York , in press). 22. Yuan Q. et al. Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone. Appl. Environ. Microbiol. 63, 263–269 (1997). 23. Schier, R. et al. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J. Mol. Biol. 255, 28–43 (1996). 24. Smith, G.P. Filamentous phages as cloning vectors. Biotechnology 10, 61–83 (1988). 25. Kramer, A. & Schneider-Mergener, J. Synthesis of peptide libraries on continuous cellulose membranes. Methods Mol. Biol. 87, 25-39 (1997). 26. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning. A laboratory manual, Edn. 2. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

Counting alleles reveals a connection between chromosome 18q loss and vascular invasion Wei Zhou1, Gennaro Galizia2, Steven N. Goodman3, Katharine E. Romans4, Kenneth W. Kinzler1, Bert Vogelstein1*, Michael A. Choti5, Elizabeth A. Montgomery4 1Molecular

Genetics Laboratory, Johns Hopkins Oncology Center, Baltimore, MD 21231. 2Department of Surgical Sciences, Second University of Naples School of Medicine, Naples, Italy. 3Division of Biostatistics, Johns Hopkins Oncology Center, Baltimore, MD 21231. 4Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. 5Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. *Corresponding author ([email protected]). Received 21 July 2000; accepted 13 October 2000

The analysis of loss of heterozygosity (LOH) is perhaps the most widely used technique in cancer genetics. In primary tumors, however, the analysis of LOH is fraught with technical problems that have limited its reproducibility and interpretation. In particular, tumors are mixtures of neoplastic and nonneoplastic cells, and the DNA from the nonneoplastic cells can mask LOH. We here describe a new experimental approach, involving two components, to overcome these problems. First, a form of digital PCR1 was employed to directly count, one by one, the number of each of the two alleles in tumor samples. Second, Bayesian-type likelihood methods were used to measure the strength of the evidence for the allele distribution being different from normal. This approach imparts a rigorous statistical basis to LOH analyses, and should be able to provide more reliable information than heretofore possible in LOH studies of diverse tumor types. NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001

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TECHNICAL REPORTS The procedure used for the biochemical analysis, called “digital SNP,” is outlined in Figure 1. DNA is purified from microdissected, paraffin-embedded tumors, diluted, and robotically distributed to the wells of a 384-well plate. The contents of each well are used in a PCR, employing primers that amplify a single-nucleotide polymorphism (SNP) locus for which the patient’s normal cells are heterozygous. Following the PCR, two molecular beacons are added to the reaction, one labeled with fluorescein and the other labeled with hetrachloro-6-carboxyfluorescein (Hex). The beacons are identical except for the polymorphic base representing the SNP. The fluorescence in each well is then determined by fluorometry, and the number of alleles of each type directly determined from the fluorescence measurements. To establish the quantitative nature of the allelic proportion data obtained with such measurements, we mixed two genomic DNA samples that were each homozygous for two SNP alleles. The allelic proportions of these mixtures, as determined by digital SNP, were in excellent accord with the predicted ratios (correlation coefficient = 0.999, P < 0.001; see Supplementary Figure 1 in the Web Extras page of Nature Biotechnology Online). To illustrate this technology, we analyzed chromosome 18q in a panel of colorectal cancers. Chromosome 18q LOH has been extensively evaluated in the past, and chromosome 18q loss has been associated with a poor prognosis2–4. Molecular beacons were successfully designed for six different SNPs on chromosome 18q (see Supplementary Table 1 in the Web Extras page of Nature Biotechnology Online). The magnitude of fluorescence signals recorded for a representative SNP are shown in Figure 2. Wells containing template molecules resulted in fluorescence signals that were well above the background fluorescence (Fig. 2A, B). The ratios of fluorescence of the two molecular beacons reliably distinguished DNA templates that were homozygous for each allele (Fig. 2C). An example of the data obtained from a tumor sample and its corresponding normal tissue is shown in Figure 2D. Wells with no green or red fluorescence are uncolored, and represent wells that contained no DNA template molecules. Wells colored green had a ratio of green (excitation at 485 nm, emission at 530 nm) to red (excitation at 530 nm, emission at 585 nm) fluorescence >5, and were derived from a template molecule containing the allele with a sequence that was complementary to the fluorescein-labeled molecular beacon. Similarly, wells colored red had a ratio of green to red fluorescence 50%) exceeded the boundary value, that tumor was deemed to have LOH. Vascular invasion assessment. Routine histological sections were reviewed for the presence of vascular invasion. Blood vessels were distinguished from lymphatics by both the presence of thicker muscular walls and the presence of luminal erythrocytes as described18,19. Invasion of such vessels was identified by any of the following: (1) tumor cell thrombi within the lumen (2) tumor cells lining the endothelial surface, or (3) destruction of the vessel wall by tumor cells. All available slides from each tumor (mean 5.2, median = 5, range 1–17) were reviewed without knowledge of molecular results or outcome.

Note: Supplementary Figures 1 and 2 as well as Tables 1 and 2 can be found on the Nature Biotechnology website in Web Extras (http://biotech.nature.com/web_extras). Acknowledgments This work is supported by NIH grant CA62924 and CA43460. Under a licensing agreement between the Johns Hopkins University and EXACT Laboratories, Inc., Digital PCR technology was licensed to EXACT, and Drs. Vogelstein and Kinzler are entitled to a share of royalties received by the University from sales of the licensed technology. The terms of these arrangements are being managed by the university in accordance with its conflict of interest policies.

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