EP2356229A1

EP2356229A1 - Method for preparing high-throughput sequenceable dna from individual plaques of phages presenting peptides

Info

Publication number: EP2356229A1
Application number: EP09741232A
Authority: EP
Inventors: Bastian Budde; Amber Riblett; Sascha Plug; Jürgen BENTING
Original assignee: Bayer Technology Services GmbH
Current assignee: Bayer Intellectual Property GmbH
Priority date: 2008-11-03
Filing date: 2009-10-21
Publication date: 2011-08-17
Also published as: WO2010060507A1; DE102008055606A1; US20110257045A1

Abstract

The present invention relates to a method for DNA isolation from individual plaques of bacteriophages presenting peptides in a high-throughput PCR, wherein the PCR products obtained are sequenceable and a sample of each analyzed phage remains in a state capable of reproduction. The PCR takes place despite the presence of inhibiting components from the culture medium or the host bacteria.

Description

A method for the high-throughput preparation of sequencing-competent DNA from individual plaques of peptides presenting phages

The present invention relates to a method for DNA isolation from single plaques of peptide-presenting bacteriophages in a high-throughput PCR, wherein the PCR products obtained are sequenceable and a sample of each phage examined remains in a reproducible state. The PCR succeeds despite the presence of inhibiting components, for example, from the culture medium or the host bacteria.

Bacteriophages (short: phages) are virus-like particles that can infect bacteria and use them as specific host cells for propagation. They consist of one or more envelope proteins that include the phage genome (ssDNA or dsDNA).

An important biotechnological application for bacteriophages utilizes the direct coupling of genotype and phenotype. By genetically modifying the phage genome by incorporating appropriate oligonucleotide sequences into the DNA, peptides, protein portions or entire proteins can be coupled (fused) to phage coat proteins and thus presented on the surface of the phage. Randomisiert a part of the ligated into the DNA oligonucleotide sequences, one obtains a correspondingly large library of phages correspondingly carrying randomized peptides (English, "phage display library").

Such libraries are used, for example, to identify binding partners in which, in a repetitive selection process ("biopanning"), the library phages first interact with a substrate via their presented peptides and subsequently wash out weakly or non-binding phages ("phage display ") is used, for example, to screen protein-protein interactions. Another possible application is the identification of phages as selective, optionally self-assembling adhesion promoters. The coupling of an inorganic substrate with biological components for modifying the surface properties is known in biomimetics. From a phage library selected, short peptides presenting bacteriophages have hitherto been used, for example, to precipitate or deposit inorganic materials (WO2003 / 078451). Also, hybrid materials of an inorganic substrate and specific polypeptide ligands are used as a potential solution for changing the substrate surface. The concept of a bifunctional ligand for binding two inorganic components is addressed in the art (see, for example, Sarikaya et al., Nature Materials, 2003, 2, 577-585). The binding of cells or biomolecules to a polymer substrate by bacteriophages having bifunctional binding properties is described, for example, in WO2004 / 035612. It is possible to use bacteriophages to treat the Adhesion of a coating material (eg a paint) on surfaces to be coated (eg a polymer workpiece) to improve. It is likewise possible to use phages for the adhesion promotion of an active substance with a substrate in order, for example, to bind active substances to a target in a targeted manner, to direct active substances in a targeted manner into a body region or to achieve a time-delayed release of active substances.

In summary, there is a great need for the production, isolation and identification of phages with specific properties for a variety of applications.

Selection of phages from a phage library in which the phages present a variety of different peptides is known in the art (Sarikaya et al., Nature Materials, 2003, 2, 577-585, O'Neil and Hoess, Current Opinion in Structural Biology, 1995, 5, 443-449; Smith and Scott, Methods in Enzymology 1993, 217, 228-257, Sambrook and Russell (eds), 2001, Molecular cloning: A laboratory manual (third edition), CoId Spring Harbor Press, pages 18.115 to 18.122).

For the evolutionary selection of some phage species from a large combinatorial phage population on a substrate, a phage display library is usually exposed to a substrate so that binding of some phage can take place. Weak or non-binding phages are washed off using a wash buffer. After washing still binding phages are then peeled off (eluted) using another buffer (elution buffer). The eluted phages are propagated and re-exposed to the substrate in further rounds of panning until a population of well-binding phage accumulates. After each round of panning, the phage clones are usually singulated on agar plates in a so-called plaque assay and in a random sample a sufficiently large number of phage plaques are selected and picked up from the nutrient medium (picking). These are then separately amplified in a lengthy process in host cells, separated from the host cells and purified by repeated precipitation and concentrated to obtain a sufficient amount of DNA for the following steps. By lysis of the protein shell, the DNA of the phage clones is cleaned with subsequent precipitation and concentrated. This DNA can then be used as a template for a polymerase chain reaction (PCR) to determine its base sequence (Sanger DNA sequencing). In sequencing, the base sequence is usually determined only for that DNA segment in which the phage clones differ.

Frequently, the sample of clones examined for their sequence is on the order of 10 pieces. But if you want a more accurate statement about the enrichment of a clone or the appearance of a binding motif often requires sequencing of 50-100 phage clones.

Although high-throughput methods for the preparation of sequencing-capable DNA (sequencing templates) can be found in the prior art, these methods always require larger amounts of fermented phage suspension as the DNA source, without resorting to the time-saving and efficient PCR method. However, the recovery of the phage suspensions from the individual plaques always requires several hours of fermentation in host bacteria. Thus isolated e.g. Wilson (Biotechniques 1993, 15 (3), pp 414-422) the DNA by sodium iodide treatment of phage suspensions with subsequent ethanol precipitation or magnetic separation. Haas and Smith (Biotechniques 1993, 15 (3), pp. 422-41) prepare the DNA from phage suspensions by alkaline hydrolysis. In DE3724442 Al the DNA is purified from phage suspensions via glass fiber filters. In DE69008825T2 the inventors replace the centrifugation steps by filtration processes. Körner et al. (DNA Seq. 1994, 4 (4), pp. 253-257) use a semi-automatic cell harvester, but also require larger amounts of phage suspensions. The DNA preparation from single plaques is described in Wang et al. (Biotechniques 1995, 18 (1), pp. 130-135). However, there are no replicable phages obtained there due to the lifting method used. Vaiman (Biotechniques 2002, 33 (4), pp. 764-766) uses the PCR technique to screen large λ gene libraries, but with loss of multiplicity of individual phages and the risk of contamination by formation of "superpools".

The selection of phage species from a combinatorial phage population comprises the following steps according to the prior art:

1) incubation of a phage display library with a substrate,

2) Separation of the substrate-binding and non-binding phages,

3) determination of the sequence of the presented peptide from some of the binding phage,

4) amplification of the binding phage,

5) If necessary repeated repetition of steps 1 to 4, while the amplificate of the binding phage forms the new library,

The determination of the binding peptide sequence of the phage clones consists of the following steps: A) separation of the phages from the binding phage population obtained from step 2 on a nutrient medium hosted by host bacteria by plating out a suitable dilution of the phages,

b) Separate amplification of a sufficiently large sample of binding phage clones,

c) separation of the individual phage clones from the host cells,

d) purification of phage clones and concentration,

e) isolation of the DNA of the individual binding phage clones,

f) purification and concentration of the DNA,

g) sequencing of the DNA by a polymerase chain reaction according to Sanger,

h) Determining the Identity (Sequence) of the Binding Peptide by Translating the DNA

Sequence.

This prior art approach involves many process steps between plaque assay singulation of the phage and DNA sequencing, which are time consuming and error prone. Likewise, the number of samples that can be processed in parallel is limited. Against the background of the high importance of phages and the variety of possible uses outlined above, it would be desirable to be able to accelerate and parallelize the process of identifying phages suitable for the particular application. It would also be desirable to be able to obtain the phages in a replicable state in order to separately grow interesting phage clones after sequencing and to use them for other purposes.

Accordingly, it is an object of the prior art to provide a method for the DNA preparation of candidate phage clones from a phage population, which is less expensive and less time-consuming compared to the methods known from the prior art, the parallel processing of a larger number of samples and after sequencing allows to propagate selected phage clones in their host bacteria.

Surprisingly, a high-throughput method for DNA preparation of phages has been found, which generates a DNA capable of replication and generates DNA capable of being sequenced directly from phage plaques without the complicated steps of phage amplification and DNA isolation being necessary. Surprisingly, it was found that phages after singulation a culture medium containing host bacteria (plaque assay) can be suspended in a medium immediately after picking and fed to a lysis with subsequent optimized PCR. In each case, a replicable sample remains behind from each phage clone.

The present invention therefore relates to a method for the high-throughput preparation of DNA capable of sequencing from individual plaques of peptides presenting phages while at the same time obtaining infectious phages, comprising at least the following steps:

A separation of peptides presenting phages from a phage population on a nutrient medium containing host bacteria,

B Incubation for amplification of isolated phages until sufficient

Quantity is present

C uptake of the phages from the nutrient medium and suspension in a suitable medium,

D Lysis of a portion of the suspended phage from step C and use of this DNA-containing lysate as a template in a polymerase chain reaction (PCR).

The term "peptide" is also used synonymously for the terms "protein" or "protein part." Peptides (or proteins or protein parts) can bind bacteriophages to a substrate, whereby these peptides are gene products of the phage genome which, for example, encase the envelope A binding peptide does not have to be present on the natural form of the phage ("wild-type") but can be presented on the phage, for example, by means of molecular genetic manipulations of the phage genome. Such a binding peptide is e.g. on further peptides / proteins of the bacteriophage fused, which means that it is either N-terminal or C-terminal via a peptide bond with the other protein / peptide.

A first step of the method according to the invention is the separation of peptides presenting phages from a phage population (A). Methods for the separation of microorganisms and cells are known to those skilled in the field of microbiology. The isolation can be done, for example, by plating out a suitable dilution of the phages (the appropriate dilution can be determined empirically) on a nutrient medium containing appropriate host bacteria (eg an agar plate). In a second step of the method according to the invention, the isolated phages are amplified (eg by incubation) until a sufficient amount of phage is present (B). A sufficient amount is to be understood as meaning an amount which makes it possible to carry out the subsequent steps of the method according to the invention, in particular step (C) of the uptake of the phages. Preferably, a sufficient amount is understood to mean an amount visible to the human eye which can be absorbed by a suitable tool (eg a pipette tip).

After amplification, the phages are taken up in a third step of the method according to the invention with a suitable sterile tool from the soil (picking) and the thus obtained phage, bacteria and nutrient-containing sample (the so-called. "Agar-plug") in a for the A suitable medium is defined as preserving the infectivity of the phage therein, for which the medium must have suitable conditions for hydrophilicity and polarity of the solvent as well as pH and ionic strength an aqueous solution of approximately neutral pH (eg a buffer) and suitable salinity Suitable media are, for example, LB medium (lysogeny broth) or PBS (English phosphate-buffered saline) If the phages used are the host bacteria Confer antibiotic resistance, it is advantageous to add to the medium used this antibiotic to Avoid growth of contaminating bacteria. In a high-throughput process, picking can be performed, for example, in sterile 96-well

Plates happen.

In the subsequent PCR reaction (D), many components contained in the phage plug (e.g., nutrient medium, agar, cell debris) normally inhibit, but it has surprisingly been found that a sequenceable PCR product results under the following conditions:

1. Picking the agar plaque from the culture medium is done with a suitable sterile tool. A suitable tool is defined by the fact that you can transfer the plaque completely from the nutrient medium in a working vessel, without much of the other nutrient medium are dissolved out with. A suitable tool is e.g. a tubular instrument having an inner diameter equal to the outer diameter of the

Agar plaques, with which the agar plaque can be gouged. In a preferred embodiment, a sterile disposable pipette tip or a Pasteur pipette is used, by means of which the plaque is punched out and then the agar plug is blown out into the medium with a pipette. 2. The picked phage clone is suspended in the medium and then exposed to an energy input, preferably ultrasound treatment. Also possible are vertexes as well as mechanical crushing of the agar plug. These procedures apparently promote the passage of phage from the culture medium into the medium.

3. The volume of the medium should exceed the volume of the agar plug by a multiple, preferably by a factor of 2 to 10. Preferably, a volume is chosen in which the agar plug is completely covered in the vessel used. In a preferred embodiment, an agar plug picked with a disposable pipette tip is suspended in a well of a 96-well plate in 20 to 100 μl of medium.

4. The suspension must be freshly processed. Preferably, the lysis of the clone is carried out with subsequent PCR within 16 hours, more preferably within 8 hours. If one day has elapsed between the cloning of the clone and the PCR reaction, there may be errors in the amplification of the template DNA. Apparently, a sufficiently large amount diffuses more inhibitory in the course of this period

Substances from the agar plug into the medium to impede the amplification of the template DNA.

From the suspension thus obtained, a small portion is treated with a lysis buffer and subjected to a lysis step to expose the DNA of the phage. The larger part of the phage suspension can remain in the 96Well plates and is thus available for subsequent targeted amplifications of individual interesting phage clones. The lysis buffer contains a buffer substance in aqueous solution at a slightly alkaline pH, a surfactant and a complexing agent. The buffer substance is preferably used in a pH range> 8 and <9, a non-denaturing, nonionic substance as surfactant and a chelating agent which complexes divalent metal ions. Tris, Tricin, Bicin or Taps at a pH of 8.3 are particularly preferred, as surfactant an octylphenol ethoxylate (eg Triton X-100 or Nonidet-P40) or a polysorbate (eg Tween®-20) as well as complexing agent EDTA, EGTA, citrate, oxalate or tartrate used. In a preferred embodiment, 10 mM Tris-HCl (pH 8.3), 1% (w / v) Triton X-100 and 10 mM EDTA are used. The Lyseschritt comprises heating the suspension to a temperature> 40 ⁰ C and <100 ⁰ C, preferably to a temperature> 80 ⁰ C and <98 ⁰ C, more preferably to a temperature> 93 ⁰ C and <97 ⁰ C.

Subsequently, the suspension is fed to an optimized PCR (D). Surprisingly, it was found that despite the presence of inhibitory suspension components variable region of the phage clone DNA can be amplified without error if both the conditions described above in the steps of suspension and lysis are observed as well as in the PCR reaction, an amount of dimethyl sulfoxide (DMSO) is present. According to the invention, an amount of between 0.1 and 50% by weight of DMSO is added to the PCR buffer. Preference is given to the PCR buffer between 1 and 30% DMSO, more preferably between 5 and 15% DMSO added. In a preferred embodiment about 10% DMSO are used.

Usually, a separation of the template DNA from other components is necessary before a PCR can be carried out, since the other components can interfere with or even prevent the replication of the template DNA. Surprisingly, it has been found that the isolation, purification and concentration of the phage DNA can be dispensed with by the conditions described above. Ingredients such as nutrient medium, agar and residues of the host cells surprisingly show no inhibitory influence on the PCR in the method according to the invention. This makes it possible to perform the DNA preparation process in a shorter time, with less effort, at a lower cost and reduced error rate with a larger number of samples.

Sequencing of the phage DNA, e.g. connect to Sanger. The determined sequence of the variable region of the phage clone DNA allows the conclusion to the amino acid sequence of the binding peptide. The identified DNA sequence can be used to genetically engineer other phage to present the same bindable peptides as fusion proteins.

In the event that the phages differ only in the binding peptide, preferably only the part of the phage DNA responsible for the coding of the variable peptides is amplified by PCR. This is done by choosing two suitable primers that bind to binding sites just before or just after the variable region of the phage DNA. Preferably, the primers are selected such that the resulting in the PCR amplicon has a size of> 60 and <500 bp.

The method of the invention is generally accessible to all phages that can form a plaque. It is preferably used for DNA preparation from clones of phage display libraries.

In a particular embodiment, the method according to the invention is preferably suitable as a DNA preparation process for phages which present at least one sort of randomized peptides fused to their coat proteins, which have a length of from> 1 amino acids to <100 Amino acids, preferably> 5 amino acids to <50 amino acids, more preferably> 6 amino acids to <20 amino acids. The peptides can be linear but also loop-shaped. Peptides with such a number of amino acids can be well fused to the phage proteins by conventional molecular biological methods and are suitable for binding to substrates. Examples of such peptides are 7-amino acid linear peptides, 12-amino acid linear peptides, or 7-amino acid loop peptides, which are framed by a disulfide bridge between two cysteine residues. Such short-chain binding peptides have the advantage that the DNA of the various phages in the considered phage population is largely identical and has only differences in the regions of the DNA which encodes the binding peptides. Thus, the PCR can be targeted on short DNA sections (> 60 and <500 bases), which has the advantage that the same primer and the same PCR conditions can be used for all the picked clones.

The erfmdungsgemäße method is suitable in a particular embodiment preferably as a DNA preparation process for phages of type Ml 3. These phages are well changeable and readily available (for example, New England Biolabs GmbH, Frankfurt, Germany) and readily reproducible in culture. They have as gene products the proteins gpiπ, gpVI, gpVII, gpVIH and gpIX. At least one additional short peptide may be fused to the protein gpIII or gpVTQ. The extension of these proteins with additional peptides, ie, the fusion, is well feasible on these proteins.

Advantageously, commercially available M13 phage display libraries are used, comprising a randomized peptide as a gpHI fusion protein, in particular as a linear peptide with a length of 7 amino acids, as a linear peptide with a length of 12 amino acids, or as a loop-shaped peptide with 7 amino acids , which are framed by a disulfide bridge between two cysteine residues (trade names Ph.D.-7 ™, Ph.D.-12 ™ and Ph.D.-C7C ™, respectively).

In a preferred embodiment, the method according to the invention is part of a selection process for substrate-binding phage species from a library presenting randomized peptides. In the context of the invention, a binding phage has the property that it can bind to at least one substrate via a peptide which has been fused to a phage-clad protein. The person skilled in the art is aware of this technique as a "phage display".

The present invention will be explained in more detail below with reference to figures and examples, but without restricting it to these.

Show it:

FIG. 1 shows the result of a PCR which was carried out with differently treated M13 phage plaques as DNA source. The PCR products were loaded on a 1.2% (w / v) agarose gel and electrophoresed. The agarose gel was stained with ethidium bromide and photographed under UV light. 1: DNA size standard, 2: complete, intact phage plaque, 3: phage plaque suspended in LB medium according to the invention, 4: positive control (purified phage DNA), 5: negative control (without DNA template). The phage plaque as a DNA source (lane 3) suspended in LB medium according to the invention gives a qualitatively equivalent product as the purified phage DNA (lane 4). The complete plaque can also be used successfully as a DNA source (lane 1), but no intact phage remain from this sample for later amplification. Despite the high number of 40 PCR cycles, no artifacts are found in the negative control (lane 5).

FIG. 2 shows the result of a PCR performed with M13 phage plaques suspended in LB medium as DNA source. The PCR products were loaded on a 1.2% (w / v) agarose gel and subjected to electrophoresis. The agarose gel was stained with ethidium bromide and photographed under UV light. 1: DNA size standard, 2-9: plaques of different phage clones, 10: positive control (purified phage DNA), 11: negative control (without DNA template). The 334 bp PCR products can be clearly recognized in the samples of good quality (lanes 3-9) and in the positive control (lane 10). The lower running phage clone DNA in lane 2 could be identified in the subsequent sequencing as phage without insert (equivalent to M 13KE without library insert). The negative control (lane 11) shows no artifacts as expected.

Figure 3 shows a typical elution profile of DNA sequencing performed on the primer "-96gIII" (5'-CCCTCATAGTTAGCGTAACG ^ ', see example) on a PCR product of a phage plaque according to the invention The phage clone is derived from a Ph .D.-12 ™ M13 Phage Display Library The phage clone variable DNA region of interest (position 89-124) has high sequence quality.

Example: DNA isolation of M13 phage clones. obtained by panning a combinatorial phage display library (Ph.D.-12 ™, New England Biolabs) on a polyurethane substrate.

Polyurethane substrate was made from mixing equivalent amounts of Desmophen® 670 BA and Desmodur® N3300 (Bayer MaterialScience AG), the curing was carried out for 16 h at room temperature. 20 mg of the substrate were for 10 min in Tris-buffered saline (TBS, consisting of 50 mmol / 1 Tris-HCl pH 7.5, 150 mmol / 1 NaCl) and equilibrated for 60 min with 4 * 10 ¹⁰ pfu (10 ul of the original Library) in 1 ml of TBS at room temperature. The substrate was washed ten times with 10 ml each of TBST (TBS plus 0.1 vol% Tween-20) (by short vortexing, five minutes rotation plus 5 seconds ultrasonic bath). The first elution was carried out in acid by immersing the substrate in 1 ml of 0.1 mol / 1 glycine pH 2.5 for 10 s with subsequent neutralization of the substrate in 1 ml of 0.1 mol / 1 Tris-HCl pH 8 for 1 min. The first elution solution was neutralized by adding 200 μl of 1 mol / l Tris-HCl pH 8. The second elution was carried out in basic by immersing the substrate in 1 ml of 0.1 mol / l triethylamine pH 11.5 for 1 min, followed by neutralization of the substrate in 1 ml of 0.1 mol / l Tris-HCl pH 7.5 for 1 minute The second elution solution was neutralized by adding 200 μl of 1 mol / l Tris-HCl pH 7.5. The substrate was then stored in TBST to observe time-release effects of non-eluted phage.

The elution fraction was then subjected to a plaque assay. For this purpose, bacteria of strain E. coli K12 ER2738 (New England Biolabs) on an LB-Tet agar plate (15 g / l agar, 10 g / l bacto-tryptone, 5 g / l yeast extract, 5 g / l NaCl, 20 mg / 1 tetracycline) and incubated overnight at 37 ⁰ C. Ten ml of LB-Tet medium (10 g / l bacto-tryptone, 5 g / l yeast extract, 5 g / l NaCl, 20 mg / 1 tetracycline) were inoculated with a single colony ER2738 and up to an OD _60O of 0.4 at 37 ⁰ C shaken. 400 μl of this bacterial suspension were mixed with 10 μl undiluted elution solution, pipetted into 3 ml molten LB agar top (7 g / l agar, 10 g / l bacto tryptone, 5 g / l yeast extract, 5 g / l NaCl), vortex and on an LB-IPTG / X-gal plate (15 g / l agar, 10 g / l bacto-tryptone, 5 g / l yeast extract, 5 g / l NaCl, 1.25 mg / l isopropyl-β-D thiogalactopyranoside, 1 mg / l 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside). The plate was incubated at 37 ^° C. overnight. Each of the resulting blue plaques represents a phage clone.

80 blue, well separated plaques were punched out of the agar plate with sterile 1000 .mu.l pipette tips and transported by means of a 1000 .mu.l pipette into one well of a 96-well sterile plate, in each of which 100 .mu.l LB-Tet medium were submitted. The 96-well plate was sealed with PCR strips and sonicated for 1 min in an ultrasonic bath. 5 μl each of the phage clone suspension thus obtained were pipetted immediately thereafter into one well of a new 96 well plate in which 24 μl lysis buffer (10 mM Tris-HCl pH 8.3, 10 mM Na ₂ -EDTA pH 8.0, 1% (w / v) Triton X-100). 5 μl of H ₂ O were used as negative control in a further two wells of the same 96% plate, or a mixture of 2 μl of H ₂ O and 3 μl of purified M13 phage DNA from a clone of the phage display library Ph.D. 12 ™ (New England Biolabs) pipetted.

The phage DNA for the positive control was isolated and purified in the following manner: 2 ml of LB-Tet medium were inoculated with 100 μl of an E. coli ER2738 bacterial suspension (see above) and a blue plaque from a plaque assay and incubated for 4.5 h shaken at 37 ⁰ C. The culture was centrifuged for 10 min at 4500 xg and 4 ⁰ C and the supernatant then centrifuged again. 1 ml of the supernatant was mixed with 500 ul PEG / NaCl solution and incubated for 2 h at 4 ⁰ C. The phages were 15 min at 14,000 xg and 4 ⁰ C and centrifuged in 100 ul of 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 4 M NaI resuspended. The DNA was precipitated by adding 250 ul of ethanol and 10 min at 14,000 × g for 15 min and 20 ⁰ C by centrifugation. The pellet was washed with 70% ethanol, 1 min at 14,000 xg and 20 ⁰ C centrifuged, dried and resuspended in 30 ul 10 mM Tris-HCl pH 8.0 resuspended.

The 96-well plate was sealed with PCR strips. The lysis of the phage was carried out by heating the plate for 20 min at 95 ⁰ C in a thermocycler. Subsequently, the PCR was performed. For this purpose, 26 μl of PCR master mix, which had the following composition, were pipetted into each well:

The PCR primers have the following sequence:

Primer Reverse (Gen YS): -96gEI 5 '-citocatagTTAGCGT AACG-3'

Primer Forward (Gen Vm): M13KE_F1 5'-GATGGTTGTTGTCATTGTCG ^ '

The primers in the case of the Ph.D.-12 ™ phage library used, which is based on the vector M13KE, include a region of 334 bp long containing the 36 bp variable sequence of each phage clone. The PCR cycle was performed in a thermocycler with the following conditions: 1st 30 sec 95 ⁰ C

2. 30 sec 52.2 ⁰ C

3. 30 sec 72 ⁰ C

4. 39 repetitions of steps 1-3 5. 5 min 72 ⁰ C

The PCR products were separated in a 1.2% agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 50 mM Na ₂ -EDTA pH 8.0). In this way, a sample of 8 samples was analyzed for their quality (Figure 2).

Per 10 ul of each PCR product was pipetted into a new 96 well plate and dried for 3 hrs in an oven at 50 ⁰ C. Subsequently, the plate with a self-adhesive

Aluminum foil sealed and sent to a DNA sequencing service provider (Eurofms MWG Operon). There, the samples were sequenced with the primer -96giπ by the dideoxy method (see FIG. 3). The resulting DNA sequence of each sample represents the

Anticodon strand of the template and was first rewritten in the complementary sequence. The variable region amino acid sequence could then be analyzed using the reduced genetic code (Ph.D.-12 ™ Phage Display Peptide Library Kit Instruction Manual, Ph.D.

Version 2.7 (2006), New England Biolabs).

Claims

claims

A method for the high-throughput preparation of sequencing-competent DNA from individual plaques of peptides presenting phages while simultaneously receiving infectious phages comprising at least the following steps:

B incubation for the amplification of the isolated phages,

C uptake of phages from the nutrient medium and suspension in a medium

2. The method according to claim 1, characterized in that in step C, the picking of the phage plaque is carried out of the culture medium with a sterile tool, by means of which the plaque punched out of the agar plate and then the agar plug thus obtained is transferred into the medium ,

3. The method according to claim 2, characterized in that the suspension in step C with energy input, preferably ultrasonic treatment.

4. The method according to claim 3, characterized in that the volume of the medium is at least twice as large as the volume of the agar plug and that the agar plug is completely covered in the vessel used for the suspension.

5. The method according to claim 4, characterized in that the lysis takes place in step D within a maximum of 8 hours following step C.

6. The method according to claim 5, characterized in that the lysis in step D is carried out by adding an aqueous lysis buffer comprising at least one buffer substance, a surfactant and a complexing agent.

7. The method according to any one of claims 1 to 6, characterized in that the lysis in step D comprises the heating to a temperature between 80 ⁰ C and 98 ° C.

8. The method according to any one of claims 1 to 7, characterized in that in step D, an amount between 1 and 30 wt .-% DMSO is supplied to the PCR.

9. Use of a method according to any one of claims 1 to 8 as part of a selection process for binding phage from a phage display library.