Method for the spatial arrangement of sample fragments for amplification and immobilization for further derivatizations

Abstract

The invention relates to a method for performing a biochemical or chemical reaction for an isolated, spatially separated amplification of sample fragments during a simultaneous immobilization and spatial arrangement of the sample fragments and reaction products, the amplification products, on one or more suitable solid phases for subsequent derivatizations.

Claims

1. A method for amplifying more than one sample fragment, comprising: a) supplying a solid phase with a microstructure, wherein the microstructure subdivides the solid phase so that several compartments are formed, and wherein capture molecules are attached to surfaces in the compartments, b) introducing exactly one type of sample fragment into each of the several compartments, wherein each of the sample fragments has an adapters on one or both ends, wherein the adapters have a uniform sequence with a length of 1 to 500 nucleotides and are primers or have a binding site for RNA polymerase or a cell free expression system, c) introducing a reaction mixture for amplification into the several compartments, d) separating and/or covering the individual compartments to obtain sealed compartments, and e) amplifying each sample fragment of said sample fragments independently in the corresponding sealed compartment to obtain an amplification product for each of the sample fragments in each of the compartments, f) wherein the amplification products generated by the amplifying in (e) are immobilized on the capture molecules attached to the surfaces in each of the compartments and are subsequently analyzed, sequenced and/or derivatized.

2. The method according to claim 1, wherein the capture molecules are primers.

3. The method according to claim 1, wherein the amplification products are immobilized on the capture molecules via covalent bonds.

4. The method according to claim 1, wherein the compartments are cavities.

5. The method according to claim 1, wherein the introducing of the sample fragments and the introducing of the reaction mixture take place in one step.

6. The method according to claim 1, wherein the reaction mixture and/or the sample fragments is/are added to the compartments via centrifugation, vacuum, atomization, immersion, dip coating and/or painting the surfaces of the solid phase.

7. The method according to claim 1, wherein the surfaces of the solid phase are made of semiconducting, amorphous, crystalline and/or fibrous materials.

8. The method according to claim 1, wherein the surfaces have hydrophilic and/or hydrophobic regions and/or are structured in one or more layers as a structured surface.

9. The method according to claim 1, wherein the covering and/or separating of the compartments include oil, electric forces, phase transitions, inert regions and/or covers.

10. The method according to claim 1, wherein the capture molecules have a spacer molecule and/or comprise a chemical coupling group at one or more ends.

11. The method according to claim 1, wherein the capture molecules have a length of 1 to 1000 individual molecules.

12. The method according to claim 1, wherein the capture molecules are immobilized either directly or indirectly on the solid phase.

13. The method according to claim 1, wherein the cover comprises capture molecules.

14. The method according to claim 1, wherein the capture molecules comprise at least one binding site for sample fragments and/or amplification products.

15. The method according to claim 1, wherein the capture molecules and/or amplification products have a photoactivatable cleavage group.

16. The method according to claim 1, wherein the capture molecules have a proton binding site for an RNA polymerase or a cell-free expression system.

17. The method according to claim 1, wherein the sample fragments are nucleic acids, preferably DNA or RNA, proteins, antibodies, synthetic molecules, organic molecules and/or natural substances or have been released from cells or natural substances introduced into the compartments.

18. The method according to claim 1, wherein the sample fragments are amplified directly or indirectly via an incorporated label from DNA or RNA.

19. The method according to claim 1, wherein the sample fragments have a length of 1 to 10.sup.10 individual molecules in single-stranded or double-stranded form.

20. The method according to claim 1, wherein amplification in (e) is selected in particular from the group consisting of PCR, RPA, RCA, SDA, NASBA and LAMP.

21. The method according to claim 1, wherein the reaction mixture comprises reaction-enhancing components.

22. The method according to claim 1, wherein said amplifying does not involve an emPCR.

23. The method according to claim 2, wherein the primers are primers that are used for the amplifying in (e).

24. The method according to claim 5, wherein the sample fragments are mixed with the reaction mixture before the introducing.

25. The method according to claim 21, wherein the reaction-enhancing components are BSA, Tween and/or agarose.

26. The method of claim 11, wherein the individual molecules are bases of a DNA molecule.

Description

EXAMPLES

(1) The invention will now be described on the basis of an exemplary embodiment and the accompanying drawings. The examples and illustrations are preferred embodiment variants which do not restrict the invention. The advantages that are described have also been demonstrated for the other embodiment variants mentioned and apply not only to the concrete example. The figures show:

(2) FIG. 1 Detail of a multiwell array

(3) FIG. 2 Preferred geometric embodiments of cavities

(4) FIG. 3 Additional geometric embodiments of cavities

(5) FIG. 4 Preferred capture molecules with their orientation

(6) FIG. 5 Preferred adaptors

(7) FIG. 6 Preferred device

(8) FIG. 7 Additional preferred device

(9) FIG. 8 Preferred evaluation

(10) FIG. 9 Exemplary experimental results of a solid phase PCR

(11) FIG. 10 Exemplary experimental results with signals of filled cavities

(12) FIG. 11 Cover of multiwell array after the reaction 30

(13) FIG. 1 shows a schematic detail of a multiwell array having a few cavities. All the cavities are coated with capture molecules. An amplification mix with sample fragments is introduced (at the left) and the cavities are closed. After amplification, the respective cavities are coated with amplification products (at the right).

(14) FIG. 2 shows one possible diagram of the different geometric embodiments for creating the compartmentalization for the subsequent amplification. The filled basic state is shown along with how the individual regions are shielded with respect to one another. The main interpretation is cavities (A) which are sealed with a cover. However, the cavities may also be separated from one another with oil (B), fields, in particular electric fields or by temperature (C) or a phase transition material (D).

(15) FIG. 3, like FIG. 2, shows one possible diagram of the different geometric design shapes for creating the compartmentalization for the subsequent amplification. Additional interpretations include all the structures capable of digital PCR, which are additionally coated with capture molecules (E). The sample fragments are first brought to a planar surface with capture molecules in an inverted design (F) and then are covered with a multiwell array. This is an inversion of a filling process, but it has the advantage that samples can also be enriched with highly diluted sample fragments on the planar surface. Direct filling thus could not be performed because of the excessive dilution and/or too few cavities could be filled with a sample fragment. The cavity is formed at the moment of closure. Version F may also be used to copy DNA from microarrays into a multiwell array.

(16) FIG. 4 shows different possibilities for capture molecules with their orientation.

(17) FIG. 5 shows possible embodiments of the adapters.

(18) FIG. 6 shows a possible device/cassette for amplification and/or sequencing according to the invention.

(19) FIG. 7 shows an exploded diagram of a preferred device for the reaction of the multiwell array (A) in the recess of a cup holder (B), cover (C), 3 mm thick elastic ram (D), clamping cover (E). To introduce the amplification mix into the array by centrifugation, parts C through D are replaced by a filling device which reaches a 150 m tall microfluidic chamber.

(20) FIG. 8 shows one possible evaluation of a reaction in a multiwell array with a well volume of 19 picoliters. Figures (B) through (E) show the scans of pigmented covers after the reaction. Figure (A) shows a micrograph of a multiwell array. Figure (B) shows a specificity check. In this case, a capture molecule that is not complementary to the sequence of the DNA fragment has been immobilized, so no positive signals are generated. Figure (C) shows a no-DNA fragment check. Here again, no signals are generated. This result shows that a signal is also generated in the case of the presence of a DNA fragment. Figure (D) shows the signals of a cover in which hybridization has been used to visualize the reaction. This result shows that the correct sequence of the DNA fragments was in fact also bound to the capture molecules. In Figure (E) the surface-bound PCR product was bound by coupling streptavidine-Cy5 to the biotin molecules incorporated during the reaction, and the reaction was visualized.

(21) FIG. 9 shows one possible implementation of a reaction in 110,000 19-picoliter wells. In 99.18% of all wells (109,101 signals), the PCR products were bound to the capture molecules by means of the mechanism of solid-phase PCR. This result shows that with this system, the reaction can even be performed in areas of >1 cm.sup.2.

(22) FIG. 10 shows, for example, a test for which DNA fragments of the length 100 bp, 346 bp and 1513 bp (base pairs) were used and stained with streptavidine-Cy5 after the reaction. The number of DNA fragments per well was calculated. The graph shows the number of positive signals measured by the totality of all the filled cavities (total of 10,000 wells, area 10 mm.sup.2) as a function of the length of the DNA fragments. For the 100 bp DNA fragment, 95.31.8% positive signals were counted, for the 346 bp DNA fragment 92.11.9% positive signals were counted and for the 1513 bp DNA fragment 90.93.5% positive signals were counted. This shows that with this system it is also possible to amplify and immobilize DNA fragments >>1000 bp, which is an interesting feature in comparison with the limitations of emPCR.

(23) FIG. 11 shows, for example, a cover pigmented with streptavidine-Cy5 after a reaction in a multiwell array. The amplification mix contained 0.025 DNA fragments per well. The cover shows a pattern of discrete signals originating from wells in which a DNA fragment was present. This shows that a PCR product based on a single DNA fragment can be generated and, while retaining the spatial distribution, bound to immobilized capture molecules and visualized.

Example 1

(24) Essential Sequence of the Method According to the Invention

(25) In the first step a reaction mixture containing a defined amount of sample fragments is used. The concentration of the sample fragments is usually adjusted so that there is less than one sample fragment per volume of a cavity. The mixture is then introduced into a multiwell array whose surface is coated with capture molecules. By closing (covering) the microcavities, these are isolated from one another, so that an independent amplification reaction can take place in each cavity. The amplification products are usually bound by an adapter to the capture molecules located at the surface. Thus the inside of each cavity into which a sample fragment has been introduced is coated with the corresponding amplification products. The result is a spatially clearly defined and mutually delimited arrangement of amplification products which corresponds to the original distribution of the individual sample fragments.

Example 2

(26) According to the following procedure, the capture molecules are applied to the cover, in particular the hybrid cover, which may consist of very thin layer (approx. 5 m) PDMS on a glass carrier. Preferably there is first a treatment with an oxygen plasma (40 kHz generator, 100 watt power, 1 minute treatment duration) to generate hydroxide groups. In the next step, the aminosilane APTES can be bound to this (5% 3-aminopropyl-triethoxysilane (APTES), 5% DI water, 90% ethanol, reaction overnight at room temperature). Then the homobifunctional linker 1,4-phenylene diisothiocyanate (PDITC) is preferably bound to the amino groups now at the surface (0.15 g PDITC, 5 mL pyridine, 45 mL dimethyl formamide (DMF)). Next a modified ssDNA sequence can be bound as a capture molecule in particular (in 150 mM sodium phosphate buffer, pH 8.3, reaction time: overnight at room temperature). This contains in particular an amino linker at the 5 end, followed by a six-part carbon chain (C6), followed by 10 thymines. Then the actual DNA sequence of the DNA capture molecule follows. The amplification mix preferably contains 30 U HotStar Taq Plus, 1 reaction buffer, 1.5 mM MgCl.sub.2, 300 M of each dATP, dGTP, dCTP, 225 M dTTP, 75 M biotin-dUTP, 0.5% BSA, 0.05% Tween 80, 0.125 M forward primer 5-CTG AGC GGG CTG GCA-3 (SEQ ID NO: 1) and 1000 M reverse primer 5-GCC TCC CTC GCG CCA TCA G-3 (SEQ ID NO: 2) and between 0 and 20 copies of a DNA fragment (pTYB1 plasmid).

(27) The aforementioned components are topped off with high purity DNAse/RNAse-free double-distilled water to a total of 16 L. The reaction mixture is stored on ice until the multiwell arrays have been filled. A filling device for bringing the amplification mix into the array is partially identical to that from FIG. 7. The multiwell array is preferably cut out of the 1313 mm.sup.2 pieces into a Pico Titer Plate (Roche Diagnostics) (FIG. 7A).

(28) The array is placed in the recess in the filling device (FIG. 7B), whereupon a 500 m thick structured rubber lip is preferably placed there. Next, a microstructured filling cover, for example, can be secured on top of that using two M3 screws, each filling cover having an inlet and an outlet. Next 16 L amplification mix can be introduced into the chamber. The entire filling device is placed on an oscillating rotor of a centrifuge (Multifuge 3SR Plus, Heraeus, VWR, Bruchsal, Germany) and centrifuged for 2 minutes at 2200 rpm.

(29) After centrifugation, the filled array is covered with a cover coated with capture molecules (FIG. 7C). Then a rubber stamp is placed on top (FIG. 7D) and next a clamping cover (FIG. 7E), which is then screwed in place using two M3 screws. The reaction device is preferably stored at room temperature (1t10 minutes) before transferring the mixture to a Slidecycler (peqStar in situ, PEQLAB Biotechnologie). After the amplification reaction, in particular PCR, the device is dismantled and all the parts, except for the cover, are decontaminated for at least 1 hour, preferably in DNA Exitus Plus (AppliChem). The surface-bound product, preferably the PCR product, can then be detected by reaction with streptavidine-Cy5 in particular (5 g.Math.mL.sup.1 strept-avidine-Cy5 in 100 mM sodium phosphate buffer (disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate monohydrate, pH=7.2, 0.1% Tween 80, for 5 minutes at room temperature) or a hybridization reaction (0.1 M of the hybridization probe in 5SSC, 50% formamide 0.1%, reaction time: 12 h516 h at 42 C.)).

(30) Washed covers can be scanned in the InnoScan 710 scanner from Innopsys, for example. Results of such a reaction are shown in FIGS. 8-11 as an example. The PCR product here was bound to the capture molecules on a cover and then visualized by the respective method. Twenty molecules per well were introduced into the array in all reactions, except in FIG. 11.

LIST OF REFERENCE NUMERALS

(31) 4 Sample fragment 5 Capture molecule 6 Hydrophobic surface 7 Hydrophilic surface 8 Surface 1 9 Surface 2 10 Aqueous phase 11 Oil phase 12 Cover 13 Oil 14 Force field 15 Phase transition.fwdarw.solid 19 Valve circuit 20 Capture molecule 21 Additional nucleic acid sequence 22 Adaptor 1 23 Adaptor 2 24 Sequence 1 25 Sequence 2