Method, substrate and kit for one-pot one-step assembly of DNA molecules

Abstract

A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, and a substrate and a kit for this purpose. A simple and cost-effective assembly method for DNA molecules. A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule is provided, wherein the two or more DNA molecules to be assembled are brought together in dry form with a suitable reaction medium on at least one substrate present in a reaction vessel.

Claims

1. A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, comprising providing two or more DNA molecules to be assembled, wherein the two or more DNA molecules to be assembled are in dry form on or in at least one substrate selected from the group consisting of a piece of filter paper, a cellulose particle, and a tablet, wherein the two or more DNA molecules are each present on or in the at least one substrate in a quantity sufficient for assembly, and bringing the two or more DNA molecules to be assembled together with a suitable reaction medium in a reaction vessel such that an assembly of the two or more DNA molecules is brought about.

2. The method according to claim 1, wherein each of the two or more DNA molecules present on or in the substrate is flanked by type IIs restriction endonuclease restriction sites with opposite orientation.

3. The method according to claim 1, wherein the two or more DNA molecules are each present on or in a separate substrate in dried form, and are brought together with the reaction medium in the reaction vessel.

4. The method according to claim 1, wherein the two or more DNA molecules are brought together in a reaction medium which contains a) a type IIs restriction endonuclease and a DNA ligase or b) an exonuclease, a DNA polymerase and a DNA ligase.

5. A kit for use in a method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, the kit comprising at least one substrate selected from the group consisting of a piece of filter paper, a cellulose particle, and a tablet, the at least one substrate comprising thereon or therein at least one of the two or more DNA molecules in dry form, which at least one of the two or more DNA molecules can be assembled with the other of the two or more DNA molecules, wherein the at least one substrate with the at least one of the two or more DNA molecules is formed and dimensioned such that it can be arranged individually as a whole and unchanged in a micro reaction vessel, and wherein the at least one substrate comprises the at least one of the two or more DNA molecules in a quantity sufficient for an assembly reaction, and wherein the two or more DNA molecules are flanked in each case by type IIs restriction endonuclease restriction sites with opposite orientation.

6. The kit according to claim 5, wherein each of the two or more DNA molecules is present on or in a separate substrate in a quantity sufficient for the assembly.

7. The kit according to claim 5, further comprising a) a type IIs restriction endonuclease and a DNA ligase or b) an exonuclease, a DNA polymerase and a DNA ligase.

8. A method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, comprising: (a) providing, in dry form, at least one substrate selected from the group consisting of filter paper, a cellulose particle, and a tablet, having on or in the at least one substrate two or more DNA molecules to be assembled, wherein the two or more DNA molecules are each present on or in the at least one substrate in a quantity sufficient for assembly, placing the at least one substrate in a reaction vessel, adding a suitable reaction medium to the reaction vessel to provide a reaction mixture of said two or more DNA molecules and said reaction solution, and incubating the reaction mixture such that an assembly of the two or more DNA molecules is brought about, or (b) providing, in dry form, at least two substrates selected from the group consisting of a piece of filter paper, a cellulose particle, and a tablet, each of the at least two substrates having on or in each substrate at least one of said two or more DNA molecules to be assembled, wherein the two or more DNA molecules are each present on or in the at least two substrates in a quantity sufficient for assembly, placing the at least two substrates in a reaction vessel, adding a suitable reaction medium to the reaction vessel to provide a reaction mixture of said two or more DNA molecules and said reaction solution, and incubating the reaction mixture molecules such that an assembly of the two or more DNA molecules is brought about.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic depiction of an embodiment of the method according to the invention.

(2) FIG. 2 shows a schematic construction of insert and vector plasmids for use in the embodiment of the method according to the invention depicted in FIG. 1 for the case of “Golden Gate” cloning.

(3) FIG. 3 shows a further schematic construction of insert and vector plasmids for use in the embodiment of the method according to the invention depicted in FIG. 1 in case of a “Golden Gate” cloning.

DETAILED DESCRIPTION OF THE INVENTION

(4) FIG. 1 shows schematically an embodiment of the method according to the invention. Aliquots of a DNA preparation 1 of the receiver vector and a DNA preparation 2 of an insert are applied in suitable quantity to separate filter paper sheets 3. The filter paper sheets 3 loaded with DNA are dried and stored in a dry environment, for example at room temperature, until use. A filter paper sheet 3 with vector preparation and a filter paper sheet 3 with insert preparation are placed in a reaction vessel 4 and brought into contact with a reaction solution, which for example contains restriction enzyme and ligase. After incubation of the assembly reaction mixture, E. coli for example is transformed with the recombinant plasmid contained in the liquid supernatant.

EXAMPLES

Example 1

(5) 40 fmol (2.4 μL) of plasmid pICH50251 (see left-hand side of FIG. 2 for the construction; depicted sequence portions in SEQ ID NO: 1, 2) were pipetted onto a small piece of filter paper 3 (square with 2 mm edge length), which had been cut out from a larger filter paper (Sartorius, class 6, 80 g/m2 FT 3-312-070). 40 fmol (1.8 μL) plasmid pAGM6752 (see FIG. 2, right-hand sides; depicted sequence portions in SEQ ID NO: 3, 4) were pipetted onto a second filter paper piece 3 of similar size and having similar properties. The two filter paper pieces 3 were then dried at room temperature and stored in the dry state for 24 h.

(6) The two filter paper pieces 3 were placed in a 1.5 mL reaction vessel 4, and 3 μL 10× Promega ligase buffer, 1.5 μL BsaI (15 Units, NEB), 1.5 μL ligase (4.5 Units, Promega) and 24 μL water were added, to give a total reaction volume of 30 μL. The reaction vessel 4 was incubated for 1 h at 37° C., followed by 5 min at 50° C. and 5 min at 80° C.

(7) The total ligation reaction was transformed in 50 μL E. coli DH10b competent cells by means of heat shock. 500 μL of LB liquid medium were added to the cells and incubated for 45 min at 37° C. 50 μL of this transformation batch were plated on selection medium. An approximately equal number of blue and white colonies was obtained (in each case approximately 130 colonies). Four white colonies were picked and cultivated in liquid medium. Plasmid DNA was isolated therefrom, analysed by restriction digestion, and found to be corresponding to the anticipated construct.

(8) This result shows that the cloning can be performed directly from the dried DNA sample applied to a filter paper substrate, without the need to transform the vector and the insert DNA fragment back into bacteria strains for plasmid preparations.

Example 2

(9) DNA of the plasmids pICH47732 (insert; equivalent to plasmid pICH50251 in respect of the restriction positions for the “Golden Gate” assembly; construction see left-hand side of FIG. 3; depicted sequence portions in SEQ ID NO: 5, 6) and pICH42301 (vector; equivalent to the plasmid pAGM6752 in respect of the restriction positions for the “Golden Gate” assembly; see right-hand side of FIG. 3; depicted sequence portions in SEQ ID NO: 7, 8) were prepared with use of the Macherey Nagel Miniprep kit “NucleoSpin® Plasmid”. The DNA concentrations, which were measured with use of a “NanoDrop” UV-vis spectrophotometer (Thermo Fisher Scientific Inc.), were 275 ng and 172 ng per microlitre, or 89 and 82 fmol per microlitre.

(10) DNA for both plasmids was applied separately to sterile MCC beads, i.e. beads made of microcrystalline cellulose (Cellets® 700, HARKE Pharma GmbH), which were then placed in two separate reaction vessels and were subjected in each case to four separate treatments:

(11) Treatment 1: 10 μL DNA and 10 μL water were added to approximately 55 beads in a 1.5 ml reaction vessel.

(12) Treatment 2: 10 μL DNA and 10 μL of a 5% trehalose solution (sterile) were added to approximately 55 beads in a 1.5 ml reaction vessel.

(13) Treatment 3: 10 μL DNA and 10 μL of a 4% PVA solution (4% polyvinyl alcohol in 200 mM Tris-HCl, pH 8.0; sterile) were added to approximately 55 beads in a 1.5 ml reaction vessel.

(14) Treatment 4: 10 μL DNA and 10 μL of a trehalose-PVA solution (2% trehalose, 4% polyvinyl alcohol, in 200 mM Tris-HCl, pH 8.0; sterile) were added to approximately 55 beads in a 1.5 ml reaction vessel.

(15) DNA and beads of the 8 reaction vessels were air-dried overnight at room temperature. The added DNA quantity for each plasmid should be 15 to 16 fmol per bead.

(16) The next day, a bead coated with pICH47732 and a bead coated with pICH42301 (both from the same treatment) were placed in a common PCR reaction vessel. 12 μL sterile H.sub.2O, 1.5 μL 10× ligation buffer, 1 μL ligase and 0.5 μL BsaI restriction enzyme were added to this. The reaction vessel was closed, pinched between the fingers and introduced into a thermocycler with the following parameters 37° C., followed by 5 mM incubation at 50° C. and 5 min at 80° C. The supernatant was transformed by means of heat shock into competent E.-coli cells. 30 μL of the 565 μL transformation mixture were plated on LB plates containing X-Gal and Carbenicillin. Plates 1 to 4 had the following colonies:

(17) Treatment 1: 155 white colonies, estimated 23250 for the entire transformation.

(18) Treatment 2: 282 white colonies, estimated 42300 for the entire transformation.

(19) Treatment 3: 196 white colonies, estimated 29400 for the entire transformation.

(20) Treatment 4: 143 white colonies, estimated 21450 for the entire transformation.

(21) For all treatments the majority of the colonies were white and the minority were blue. DNA was extracted from two white colonies per treatment. In all cases it was found that they contained the correct construct.

(22) Cloning with use of MCC beads coated with dry DNA thus proved to be very efficient.

(23) Further experiments were carried out in order to test a longer drying time prior to cloning and to vary the composition of the DNA solution.

(24) DNA for both plasmids was introduced separately to sterile MCC beads (Cellets® 700, HARKE Pharma GmbH) into two separate reaction vessels and was subjected to the following three treatments:

(25) Treatment 5: 10 μL DNA and 10 μL of a 4% PVA solution (4% polyvinyl alcohol in 200 mM Tris-HCl, pH 8.0; sterile) were added to approximately 55 beads in a 1.5 ml reaction vessel (same composition as treatment 3).

(26) Treatment 6: 10 μL DNA, 4 μL of a sterile 4% PVA solution (4% polyvinyl alcohol in 200 mM Tris-HCl, pH 8.0; sterile) and 6 μL water were added to approximately 55 beads in a 1.5 ml reaction vessel.

(27) Treatment 7: 10 μL DNA, 2 μL of a sterile 4% PVA solution (4% polyvinyl alcohol in 200 mM Tris-HCl, pH 8.0; sterile) and 8 μL water were added to approximately 55 beads in a 1.5 ml reaction vessel.

(28) DNA and beads of the 6 reaction vessels were air-dried at room temperature.

(29) Two weeks later, cloning reactions were carried out with a bead of the insert and a bead of the vector, as described beforehand for treatments 1 to 4. The transformation was carried out as described for the previous experiment, resulting in the following number of colonies:

(30) Treatment 5: 250 white colonies, estimated 37500 for the entire transformation.

(31) Treatment 6: 181 white colonies, estimated 27150 for the entire transformation.

(32) Treatment 7: 172 white colonies, estimated 25800 for the entire transformation.

(33) These results show that the beads can be stable for at least 2 weeks at room temperature and are still suitable for efficient cloning.