阅读说明：本技术 将核酸固定于固相基质表面的方法以及从液相组合物中分离物质的方法 (Method for immobilizing nucleic acid on surface of solid phase substrate and method for separating substance from liquid phase composition ) 是由 董鸣 林至诚 于 2021-08-24 设计创作，主要内容包括：本发明涉及生物检测技术领域,具体而言,涉及一种将核酸固定于固相基质表面的方法以及从液相组合物中分离物质的方法。该方法包括：a)将含有待固定核酸的溶液附着在所述固相基质的表面；b)对所述固相基质的表面进行照射以使得所述待固定核酸与所述固相基质的表面发生光交联；其中,所述溶液中含有3M～4M的异硫氰酸胍。高浓度的异硫氰酸胍能够有利于核酸分子在光交联过程中分散更加均匀,从而可以有效提高固定后核酸的生物学活性。(The invention relates to the technical field of biological detection, in particular to a method for immobilizing nucleic acid on the surface of a solid-phase matrix and a method for separating substances from a liquid-phase composition. The method comprises the following steps: a) attaching a solution containing a nucleic acid to be immobilized to the surface of the solid phase substrate; b) irradiating the surface of the solid phase matrix to allow the nucleic acid to be immobilized to be photocrosslinked with the surface of the solid phase matrix; wherein the solution contains 3M-4M guanidinium isothiocyanate. The high-concentration guanidinium isothiocyanate can be beneficial to more uniform dispersion of nucleic acid molecules in the photocrosslinking process, so that the biological activity of the fixed nucleic acid can be effectively improved.)

1. A method for immobilizing nucleic acids on the surface of a solid substrate, comprising:

a) attaching a solution containing a nucleic acid to be immobilized to the surface of the solid phase substrate;

b) irradiating the surface of the solid phase matrix to allow the nucleic acid to be immobilized to be photocrosslinked with the surface of the solid phase matrix;

wherein the solution contains 3M-4M guanidinium isothiocyanate.

2. The method according to claim 1, wherein the nucleic acid to be immobilized is pre-linked to a single-stranded tag nucleic acid sequence and photocrosslinked to the surface of the solid phase matrix via the tag nucleic acid sequence;

optionally, the tag sequence comprises a PolyT and/or PolyC sequence;

optionally, the tag sequence is a sequence obtained by connecting 8-12 continuous T and 8-12 continuous C.

3. The method of claim 1, wherein the solution further comprises salt ions;

optionally, the salt ions are 0.3M to 0.7M sodium and/or potassium ions;

optionally, the pH of the solution is 6-8;

optionally, the solution further comprises a buffer component;

optionally, the buffer component is 15m M-25 mM Tris-HCl.

4. The method of claim 3, wherein the solvent of the solution is water;

optionally, step a) is followed by step b) and before the step b), and the step b) further comprises blending the solid phase matrix with the solution attached thereto with ethanol, wherein the volume ratio of the solution to the ethanol is 1: (0.8 to 1.2).

5. The method according to claim 1, wherein the concentration of the nucleic acid to be immobilized in the solution is 0.1. mu.M to 0.7. mu.M;

optionally, the nucleic acid to be immobilized is DNA, RNA, PNA, CNA, HNA, LNA or ANA, or a combination thereof;

optionally, the nucleic acid to be immobilized is single-stranded or double-stranded.

6. The method of claim 1, wherein the material of the solid phase matrix comprises one or more of paper, nitrocellulose, glass, magnetic materials, and plastics;

optionally, the solid phase matrix is at least one of a multi-well plate, a membrane and a microbead.

7. The method according to claim 1, wherein the light used for the photocrosslinking has a wavelength of 250nm to 260 nm.

8. The method according to any one of claims 1 to 7, wherein the nucleic acid to be immobilized is an aptamer;

optionally, the nucleic acid to be immobilized is an exosome capable of specifically recognizing and binding an exosome surface marker;

further, the surface marker is selected from AT least one of CD63, CD9, CD81, HSP70, Tsg101, EpCam, flotillin, Syntenin, Alix, HSP90, LAMP2B, LMP1, ADAM10, nicastrin, AChE, AQP2, RPL5, and a-1 AT.

9. A nucleic acid-loaded solid-phase substrate immobilized by the method of any one of claims 1 to 8.

10. A method for separating a substance from a liquid composition, characterized in that the solid phase matrix of claim 9 is incubated with a solution containing a substance to be separated so that the aptamer can bind to the substance to be separated and is immobilized on the surface of the solid phase matrix, and the substance to be separated is eluted from the surface of the solid phase matrix using the complementary strand of the aptamer;

the surface of the substance to be separated is exposed with the target of the aptamer.

11. The method of claim 10, further comprising the step of recycling the solid phase matrix, comprising:

washing the solid phase matrix with hot water at a temperature of more than or equal to 85 ℃;

and/or, washing the solid phase matrix with absolute ethanol.

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for immobilizing nucleic acid on the surface of a solid-phase matrix and a method for separating substances from a liquid-phase composition.

Background

Biochips, biological microarrays, microbeads immobilized with nucleic acids (especially aptamers) have become important tools in modern molecular biology and medicine.

The key to some of these techniques is the efficient immobilization of the nucleic acid on a support or material. The classical method of immobilizing nucleic acids on a support is the use of ultraviolet light, i.e., ultraviolet crosslinking. Church and Gilbert, in 1984, described the application of UV light at a wavelength of 254nm to cause the immobilization of DNA fragments on nylon filters (Church and Gilbert, PNAS, 1984, 81, p: 1991-. A modified version of this method is proposed by Saiki et al, PNAS, 1989,86, p: 6230-. This effect is attributed to the more efficient binding of light activated thymine bases to the membrane.

However, since such immobilization has a problem that nucleic acids are unevenly distributed during immobilization, nucleic acids are distributed and diluted even in a part of the regions, and nucleic acids are not stacked and aggregated in another part of the regions, which may affect the biological performance of nucleic acids.

Disclosure of Invention

The first aspect of the present invention relates to a method for immobilizing nucleic acids on the surface of a solid phase substrate, comprising:

attaching a solution containing a nucleic acid to be immobilized to the surface of the solid phase substrate;

irradiating the surface of the solid phase matrix to allow the nucleic acid to be immobilized to be photocrosslinked with the surface of the solid phase matrix;

wherein the solution contains 3M-4M guanidine thiocyanate (GuTc).

Optionally, the nucleic acid to be immobilized is previously connected with a single-stranded tag nucleic acid sequence and is photo-crosslinked with the surface of the solid phase matrix through the tag nucleic acid sequence.

Optionally, the tag sequence comprises a PolyT and/or PolyC sequence.

Optionally, the tag sequence is a sequence obtained by connecting 8-12 continuous T and 8-12 continuous C.

Optionally, the solution also contains salt ions;

optionally, the salt ions are 0.3M to 0.7M sodium and/or potassium ions;

optionally, the pH of the solution is 6-8.

Optionally, the solution further comprises a buffer component.

Optionally, the buffer component is 15 mM-25 mM Tris-HCl.

Optionally, the solvent of the solution is water.

Optionally, after the step a), before the step b), blending the solid phase matrix with the solution attached thereto with ethanol, wherein the volume ratio of the solution to the ethanol is 1: (0.8 to 1.2).

Optionally, the concentration of the nucleic acid to be immobilized in the solution is 0.1 μ M to 0.7 μ M.

Optionally, the nucleic acid to be immobilized is DNA, RNA, PNA, CNA, HNA, LNA or ANA, or a combination thereof.

Optionally, the nucleic acid to be immobilized is single-stranded or double-stranded.

Optionally, the material of the solid phase matrix includes one or more of paper, nitrocellulose, glass and plastic.

Optionally, the solid phase matrix is at least one of a multi-well plate, a membrane and a microbead.

Optionally, the wavelength of light used for photo-crosslinking is 250nm to 260 nm.

Optionally, the nucleic acid to be immobilized is an aptamer.

Optionally, the nucleic acid to be immobilized is an exosome capable of specifically recognizing and binding an exosome surface marker.

Optionally, the surface marker is selected from AT least one of CD63, CD9, CD81, HSP70, Tsg101, EpCam, flotillin, Syntenin, Alix, HSP90, LAMP2B, LMP1, ADAM10, nicastrin, AChE, AQP2, RPL5, and a-1 AT.

The second aspect of the present invention relates to the nucleic acid-supporting solid-phase matrix obtained by immobilization by the method described above.

A third aspect of the invention relates to a method for separating a substance from a liquid composition, comprising co-incubating a solid matrix as described above with a solution containing the substance to be separated so that the aptamer can bind to the substance to be separated and is immobilized on the surface of the solid matrix, and eluting the substance to be separated from the surface of the solid matrix with the complementary strand of the aptamer;

the surface of the substance to be separated is exposed with the target of the aptamer.

Optionally, the method further comprises the step of recycling the solid phase matrix, comprising:

washing the solid phase matrix with hot water at a temperature of more than or equal to 85 ℃;

and/or, washing the solid phase matrix with absolute ethanol.

The invention has the beneficial effects that:

the inventor surprisingly finds that the guanidine isothiocyanate with high concentration can be beneficial to more uniform dispersion of nucleic acid molecules in the photocrosslinking process, so that the biological activity of the fixed nucleic acid can be effectively improved. The method is simple and easy to operate, has obvious technical effect, and can be widely popularized and used.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a result of detecting relative exosome yield of example 1 and a comparative example using Western blotting in one example of the present invention;

FIG. 2 is an electron micrograph of exosomes isolated in one example of the present invention, A is the result of example 1, and B is the result of example 2.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The first aspect of the present invention relates to a method for immobilizing nucleic acids on the surface of a solid phase substrate, comprising:

attaching a solution containing a nucleic acid to be immobilized to the surface of the solid phase substrate;

irradiating the surface of the solid phase matrix to allow the nucleic acid to be immobilized to be photocrosslinked with the surface of the solid phase matrix;

wherein the solution contains 3M to 4M (e.g., 3.2M, 3.4M, 3.5M, 3.6M, 3.8M) of guanidinium isothiocyanate.

The term "photocrosslinking" relates to an interaction between the support material and the nucleic acid by forming a molecular interaction or bond that links the two structural elements together under the influence or drive of energy provided by the energy source light. In the context of the present invention, photocrosslinking is carried out by using light of the usual wavelength between about 200nm and about 500nm, preferably at 256nm, on the nucleic acid molecules in order to bring about the interaction between the molecules and the support material. Typically, the interaction caused between the molecule and the support material is covalent binding of the nucleic acid and the material. Photocrosslinking in the range of about 200nm to about 500nm may be performed, for example, by using near or long wavelength ultraviolet light, UVA light, or invisible light. Basically, the linkage is effected via bases of the nucleic acid molecules, for example guanine, uracil or thymine and to some extent also cytosine or adenine residues, which react with corresponding and suitable functional chemical groups on the support material, as is known to the person skilled in the art. The term "range of about 200nm to about 500 nm" refers to every single wavelength between 200nm to 500 nm. Also preferred are certain subranges thereof, e.g., subranges from 200nm to 220nm, from 220nm to 240nm, from 240nm to 250nm, from 250nm to 260nm, from 260nm to 280nm, from 300nm to 320nm, from 320nm to 340nm, from 340nm to 360nm, from 360nm to 380nm, from 380nm to 400nm, from 400nm to 420nm, from 420nm to 440nm, from 440nm to 460nm, from 460nm to 480nm, from 480nm to 500 nm. Generally, crosslinking at wavelengths between about 200nm to about 500mn can be considered non-classical uv or long wavelength crosslinking.

The wavelength of the light used can be determined primarily by the choice of lamp. For example, to establish wavelengths in the spectrum of 200nm to 500nm, a high pressure mercury ultraviolet lamp may be used. Such lamps typically emit not just one wavelength but a range of wavelengths (spectrum) as known to those of ordinary skill in the art. The term "200 nm to 500nm spectrum" may be the spectrum emitted from a high pressure mercury uv lamp. Alternatively, light may be emitted from an LED, which may have a different emission spectrum, or from any other lamp or light source known to those of ordinary skill in the art, as long as the dominant line of emission wavelengths is in the range of 200nm to 500 nm.

In some embodiments, the nucleic acid to be immobilized is pre-attached to a single-stranded tag nucleic acid sequence and photocrosslinked to the surface of the solid phase matrix via the tag nucleic acid sequence.

In some embodiments, the tag sequence comprises a PolyT and/or PolyC sequence.

In some embodiments, the tag sequence is a sequence of 8-12 consecutive T's and 8-12 consecutive C's.

In some embodiments, the tag sequence is a sequence of 9, 10, or 11 consecutive T's joined to 9, 10, or 11 consecutive C's.

In some embodiments, the tag sequence is located at the 3 'end or the 5' end of the nucleic acid to be immobilized.

In some embodiments, the solution further comprises salt ions.

In some embodiments, the salt ion is an inorganic salt ion.

In some embodiments, the salt ion is a monovalent salt ion of 0.3M to 0.7M.

In some embodiments, the salt ion is 0.3M to 0.7M sodium and/or potassium ion, preferably 0.5M, and the salt ion can be formulated in NaCl and/or KCl form.

In some embodiments, the solution has a pH of 6 to 8, such as 7.

In some embodiments, the solution further comprises a buffer component.

In some embodiments, the buffer component is Tris-HCl 15m M to 25mM (e.g., 17mM, 20mM, or 23 mM).

In some embodiments, the solvent of the solution is water.

In some embodiments, optionally, step a) is followed by step b) and further comprising blending the solid phase substrate with the solution attached thereto with ethanol, wherein the volume ratio of the solution to ethanol is 1: (0.8 to 1.2).

The addition of ethanol facilitates removal of the solvent (e.g., volatilization).

The method optionally further comprises the step of drying the solution attached to the surface of the solid phase substrate.

The drying method is selected from air drying, oven drying, lyophilizing, or their combination.

Preferably air dried and/or lyophilized.

In some embodiments, the concentration of the nucleic acid to be immobilized in the solution is 0.1. mu.M to 0.7. mu.M; for example, 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M, 0.6. mu.M.

In some embodiments, the nucleic acid to be immobilized is DNA, RNA, PNA, CNA, HNA, LNA or ANA, or a combination thereof.

The DNA may be in the form of, for example, A-DNA, B-DNA or Z-DNA. The RNA may be, for example, p-RNA (i.e., pyranyl-RNA) or a structurally modified form, such as hairpin RNA or stem-loop RNA.

In a further preferred embodiment, the nucleic acid to be immobilized as defined above may be in the form of a short oligonucleotide, a long oligonucleotide or a polynucleotide.

The term "PNA" relates to peptide nucleic acids, i.e. artificially synthesized polymers similar to DNA or RNA, which are used in biological research and medical treatment. PNA backbones are typically composed of repeating N- (2-aminoethyl) -glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are generally described as similar peptides, with the N-terminus on the first (left) side and the C-terminus on the right side.

The term "CNA" relates to a cyclic ethane amino acid nucleic acid. Furthermore, the term relates to cyclopentane nucleic acids, i.e. nucleic acid molecules comprising, for example, 2 '-deoxycarbaguanosine (2' -deoxyarbaguanosine).

The term "HNA" relates to hexitol nucleic acids, i.e. DNA analogues, which are composed of standard nucleobases and a phosphorylated 1, 5-anhydrohexitol backbone.

The term "LNA" relates to locked nucleic acids. Typically, locked nucleic acids are modified and thus no RNA nucleotides are available. The ribose moiety of the LNA nucleotide can be modified by additional bridges connecting the 2 'and 4' carbons. Such bridges lock the ribose in the 3 'endo (3' -endo) structural conformation. The locked ribose conformation enhances base stacking and backbone pre-organization. This can significantly improve the thermal stability, i.e. the melting temperature of the oligonucleotide.

The term "ANA" relates to arabinonucleic acids or derivatives thereof. In the context of the present invention, a preferred ANA derivative is 2 ' -deoxy-2 ' -fluoro-P-D-arabinonucleoside (2 ' F-ANA).

In some embodiments, the nucleic acid to be immobilized is single-stranded or double-stranded.

In some embodiments, the material of the solid phase matrix comprises one or more of paper, nitrocellulose, glass, magnetic materials, and plastics. The plastic may be nylon or polystyrene.

The magnetic material can be metal (metal simple substance or alloy), nonmetal, or composite formed by metal and nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe)₂O₃Or Fe₃O₄Magnetic nanoRice grains); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.

In some embodiments, the solid phase matrix is at least one of a multi-well plate, a membrane, a microbead.

The term "microbead" can be a sphere, a nearly sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm; preferably 150 μm or more.

In a preferred embodiment, 0.7-0.76 g of glass beads of 100-200 μm are incubated with 0.5mL of the solution.

In some embodiments, the nucleic acid to be immobilized is a naked nucleic acid or a purified nucleic acid.

In some embodiments, the nucleic acid to be immobilized is an aptamer.

In some embodiments, the nucleic acid to be immobilized is an exosome capable of specifically recognizing and binding an exosome surface marker.

In some embodiments, the surface marker is selected from AT least one of CD63, CD9, CD81, HSP70, Tsg101, EpCam, flotillin, Syntenin, Alix, HSP90, LAMP2B, LMP1, ADAM10, nicastrin, AChE, AQP2, RPL5, and a-1 AT; preferably, the surface marker is selected from more common proteins, tetraspanin (CD9, CD63, CD81), heat shock protein 70(Heat shock protein 70, HSP70), tumor susceptibility gene 101 protein (tumor susceptibility gene 101, TSG101), ALG-2-interacting protein X (ALG-2-interacting protein X, Alix) and the like, which can be used as markers of extracellular vesicles.

The second aspect of the present invention relates to the nucleic acid-supporting solid-phase matrix obtained by immobilization by the method described above.

A third aspect of the invention relates to a method for separating a substance from a liquid composition, comprising co-incubating a solid matrix as described above with a solution containing the substance to be separated so that the aptamer can bind to the substance to be separated and is immobilized on the surface of the solid matrix, and eluting the substance to be separated from the surface of the solid matrix with the complementary strand of the aptamer;

the surface of the substance to be separated is exposed with the target of the aptamer.

In some embodiments, the method further comprises the step of recycling the solid phase matrix, comprising:

washing the solid phase matrix with hot water at a temperature of more than or equal to 85 ℃;

and/or, washing the solid phase matrix with absolute ethanol.

The complementary strand is washed off by hot water, the operation is simpler, and the effect of repeated use can be achieved.

In some embodiments, the substance to be isolated is an extracellular vesicle. In the present invention, Extracellular Vesicles (EVs) are defined as a 20nm to 1000nm sized population of membrane vesicle structures, which may include exosomes (exosomes), microvesicles (microviscles), apoptotic bodies (apoptotic bodies), and the like.

In some embodiments, the substance to be isolated is in a composition selected from the group consisting of cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspirate, bronchoalveolar lavage, pleural fluid, urine, semen, follicular fluid, uterine fluid, bile, amniotic fluid, vaginal secretions, saliva, sputum, or a clarified lysate obtained from a biological tissue sample.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1

Chromatographic column production

1. 0.73g of glass beads (100 mesh or larger) were weighed and washed.

2. Mu.l of 100. mu.M CD63 aptamer (with TC-Tag at the end) was added to 498. mu.l of a chaotropic salt solution (4M guanidinium isothiocyanate C, 0.5M NaCl, 20mM Tris-HCl, pH7) with the sequence: TTTTTTTTTTCCCCCCCCCCCACCCCACCTCGCTCCCGTGACACTAATGCTA

3. The solution was added to weighed glass beads.

4. 500ul of absolute ethanol was added.

5. Mixing for 5min, sucking off liquid, spreading the glass beads on plastic tray, and air drying.

6.256nm ultraviolet irradiation glass beads (cumulative energy not less than 0.3J/cm)²) And turning over in the middle.

7. The glass beads were packed into a chromatography column.

8. The column was washed with 0.1 XSSC Buffer for 10 min.

9. The column was washed 2 times with 0.5% SDS solution.

10. The column was washed 3 times with 1 × PBS Buffer.

11. Adding PBS to immerse the filler, covering and sealing, and storing at 4 ℃.

Note:

1×PBS Buffer：

Component	Concentration
		NaCl	0.137M
KCl	0.0027M
		Na2HPO4	0.01M
KH2PO4	0.0018M

pH＝7.4

20×SSC Buffer：

Component	Concentration
		NaCl	3M
Sodium Citrate	0.3M

pH＝7.0

example 2

The difference from example 1 is that the step 4 is eliminated and the rest of the steps are the same.

Comparative example

The difference from example 1 is that guanidine isothiocyanate C was replaced with an equal amount of water.

Example 3

The chromatography columns prepared in example 1, example 2 and comparative example were used to extract exosomes:

1. 350ul of serum was added to the column and forced through the packing. Adding the eluate back to the chromatographic column, and repeatedly passing through the filler for 15 min.

2. The column was washed 3 times with PBS Buffer.

3. The CD63 aptamer complement (200ul, 10uM) was added. The sequence is as follows: TAGCATTAGTGTCACGGGAGCGAGGTGGGGTG

4. The complementary sequence solution was pressed through the packing, and the eluate was added back to the column and passed through the packing repeatedly for 15 min. Collecting the flushing liquid as a product, and detecting the yield and the purity of the product.

Repeatedly used chromatographic column

The method comprises the following steps:

1. the column packing was washed 3 times with absolute ethanol.

2. The column packing was washed 3 times with PBS.

3. Adding PBS to immerse the filler, covering and sealing, and storing at 4 ℃.

The second method comprises the following steps:

1. the column packing was repeatedly washed with hot water above 85 ℃ for at least 3 times.

2. The column packing was washed 3 times with PBS.

3. Adding PBS to immerse the filler, covering and sealing, and storing at 4 ℃.

And (4) detecting the exosome extracted by the second method. Relative exosome yields were determined by Western blotting using an anti-TSG 101 (one of the exosome-tagged proteins) antibody, and the relative exosome yields for example 1 and the comparative example are shown in figure 1.

The electron micrograph of the obtained exosome is shown in fig. 2; from the electron micrograph, the significant particle diameters of the particles of the examples 1 and 2 are in the range of 30-150nm, and the typical characteristics of the exosomes are met. No significant small particle (<30nm) impurities were found, nor large vesicle structures with diameters above 150nm were found, and therefore the purity was very high, approaching 100%. Comparative example Western blotting appeared to have extracted, but no obvious particles were visible under electron microscopy, probably because the yield was too low to successfully prepare electron microscopy samples.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.