US20070190537A1

US20070190537A1 - Solid phase synthesis

Info

Publication number: US20070190537A1
Application number: US11/459,626
Authority: US
Inventors: Joon-Won Park; Bong-Jin Hong
Original assignee: Posco Co Ltd; Pohang University of Science and Technology Foundation
Current assignee: Pohang University of Science and Technology Foundation; Posco Holdings Inc
Priority date: 2005-07-22
Filing date: 2006-07-24
Publication date: 2007-08-16
Also published as: US20080064070A1; US8647823B2

Abstract

The present invention relates to a method for synthesis of a polynucleotide on a dendron-modified surface of a substrate, and a method for stably maintaining a polynucleotide immobilized on a solid surface.

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Provisional Application No. 60/701,848 filed in the United States Patent and Trademark Office on Jul. 22, 2005, the entire content of which is incorporated hereinto by reference.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The Polymerase Chain Reaction (PCR) technology has played an important role in biotechnology, and no technique which could replace the PCR technology has yet been developed. Accordingly, the PCR technology will have an essential position in the field of biotechnology in the future.
In addition to the development of the PCR technique, the Reverse transcriptase-Polymerase Chain Reaction (RT-PCR) technique which is a method of analyzing the gene expression based on the PCR technique was invented. The RT-PCR has a higher sensitivity in detection of a small amount of RNA molecule than a Northern blot analysis, a dot blot analysis, and a nuclease protection method, and is simpler than in situ hybridization. In particular, the RT-PCT is very useful in analyzing various samples in very small amounts, and therefore, the RT-PCR method is also widely used in clinical diagnosis.
Based on these advantages of the PCR and RT-PCR methods, many attempts have been made to combine the methods with high throughput and highly parallel method such as microarray. However, unlike the PCR or RT-PCR which is typically performed in a solution, the reactions which happen on a solid surface has many disadvantages to be overcome such as non-specific adsorption, steric hindrance and electrostatic interaction between biomolecules, etc. Specifically, when the high temperature condition is required for PCR, the stability between the surface and organic thin film introduced on the surface must be maintained at a high temperature. However, the organic thin film cannot maintain its stability in a buffer solution under the high temperature when the organic thin film is introduced on a glass or gold solid surface that is widely used for immobilizing biomolecules.

SUMMARY OF THE INVENTION

The dendron-modified surface of a substrate according to the present invention can make the biomolecules immobilized on the solid surface to maintain a sufficient interval between the biomolecules, thereby reducing undesirable steric hindrance and static interaction between them. Thus, the present invention can provide the conditions required for the PCR or RT-PCR to occur under the same reaction conditions as those of the reactions in a solution state.
In addition, an organic thin film coated on the dendron-modified surface make it possible to minimize the non-specific adsorption of biomolecules and to significantly increase the thermal stability of biomolecules immobilized on the organic thin film, thereby providing improved throughput/highly parallel PCR and RT-PCR methods.
The present invention relates to a method for synthesis of polynucleotide on a dendron-modified surface of substrate, where the dendron-modified surface is obtained by chemically modifying a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface, and a terminus of the linear region of the dendron is functionalized.
In an embodiment of the present invention for synthesis of polynucleotide on the dendron-modified surface of a substrate, the polynucleotide is synthesized by reacting (a) at least a primer immobilized on the dendron; with (b) a solution comprising polymerase, dNTP or NTP, and template DNA or RNA.
In another embodiment of the present invention for synthesis of polynucleotide on the dendron-modified surface of a substrate, the polynucleotide is synthesized by reacting (a) template DNA or RNA immobilized on the dendron; with (b) a solution comprising polymerase, dNTP or NTP, and primers.
In further embodiment of the present invention for synthesis of polynucleotide on the dendron-modified surface of a substrate, the polynucleotide is synthesized by reacting (a) polymerase immobilized on the dendron; with (b) a solution comprising dNTP or NTP, primers and template DNA or RNA.
According to an embodiment of the present invention, the synthesis of polynucleotide can be performed under a high temperature or under the thermal cycles, wherein heating and cooling are repeated.
In addition, the present invention also provides a method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate under a thermal stress, wherein the substrate is chemically modified with a dendron such that a plurality of termini of the branched region of the dendron are bound to the surface, and a terminus of the linear region of the dendron is functionalized. In the method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate, the substrate is chemically modified with the dendron, after treating the substrate with a silane compound having a hydroxyl group.

BRIEF DESCRIPTION OF THE DRAWING

A more complete appreciation of the invention, and many of the attendant advantages thereof, will be readily apparent as the same becomes better understood by reference to the following detailed description when considered in conjunction with the accompanying drawing, wherein:
FIG. 1 is a schematic view showing each DNA chain extension reaction on the dendron-modified surface: (A) The DNA chain extension is performed by reacting the primers immobilized on the dendron-modified surface with free template DNA under the presence of free enzyme; (B) DNA chain extension is performed by reacting template DNA immobilized on the dendron-modified surface with the free primers under the presence of free enzyme; (C) DNA chain extension is performed by reacting the free DNA template with the free primers under the presence of enzyme immobilized on the dendron-modified surface.
FIG. 2 is a schematic view showing DNA immobilized on the dendron-modified surface, (a) the meso-spaced dendron surface of solid support, and (b) a conventional surface of solid support.
FIG. 3 shows the thermal stability of the silanated slide (A), and fluorescent image of the silanated slide (B), wherein the numerical value under each image means the number of surface treatment with PCR solution of high temperature.
FIG. 4 shows the thermal stability of Dendron/(ethylene glycol(EG)/(3-glycidoxypropyl)methyldiethoxysilane (GPDES) slide (A), and a fluorescent image of Dendron/EG/GPDES slide (B), wherein the numerical value under each image means the number of surface treatment with PCR solution of high temperature.
FIG. 5 represents the thermal stability of Dendron/TPU slide (A), and a fluorescent image of Dendron/TPU slide (B), wherein the numerical value under each image means the number of surface treatment with PCR solution of high temperature.
FIG. 6 is a schematic view showing the DNA extension reaction following the binding between the primer DNA and template DNA.
FIG. 7 is a fluorescent image of DNA microarray obtained from the chain extension by using Klenow DNA polymerase I: (A) Cy5 fluorescent image and (B) Cy3 fluorescent image.
FIG. 8 is a fluorescent image of DNA microarray obtained from the chain extension by using Tag polymerase: (A) Cy5 fluorescent image and (B) Cy3 fluorescent image.

DETAILED DESCRIPTION

The present invention relates to a method of performing PCT or RT-PCR on the dendron-modified surface, and more specifically to a thermal stability of an organic thin film introduced on the dendron-modified surface and biomolecules immobilized on the organic thin film, and to a chain extension of PCR or RT-PCR.
According to most of the amplification methods, such as PCR, RT-PCR, random priming method, and other similar DNA amplification methods, DNA or RNA can be amplified through extension process. However, most of the extension processes require enzymes having a high optimal temperature such as Taq polymerase in order to reduce the amplification time and to increase the amplification efficiency. Thus, to carry out PCR, RT-PCR, random priming method, and other similar DNA amplification methods on the solid surface, an organic thin film introduced on the surface and biomolecules immobilized on the organic thin film must be thermally stable. In addition, the dendron-modified surface provides sufficient intervals between the immobilized biomolecules, thereby allowing the immobilized biomolecules to interact smoothly with other biomolecules in the solution. Therefore, the polynucleotide synthesis such as DNA or RNA synthesis, DNA or RNA chain extension, PCR, RT-PCR, random priming nucleic acid synthesis, or the other similar polynucleotide synthesis, chain extension, or any amplification method can be carried out on the dendron-modified surface successfully and efficiently.
The dendron materials and the preparation method of the thin film on a solid surface is disclosed in US Publication No. 20050037413A1, the entire content of which is incorporated hereinto by reference.
A dendron has a plurality of termini of the branched region of the dendron which are bound to the surface, and a terminus of the linear region of the dendron which is functionalized. The dendrons are spaced at regular intervals between about 0.1 nm and about 100 nm among the linear functionalized groups. In particular, the macromolecules may be spaced at regular intervals of about 10 nm.
The terminus of the branched region may be functionalized with —COZ, —NHR, —OR′, or —PR″₃, wherein Z may be a leaving group, wherein R may be an alkyl, wherein R′ may be alkyl, aryl, or ether, and R″ may be H, alkyl, alkoxy, or O. In particular, COZ may be ester, activated ester, acid halide, activated amide, or CO-imiazoyl; R may be C₁-C₄alkyl, and R′ may be C_l-C₄alkyl. Further, in the above described substrate, the polymer may be a dendron. Still further, the linear region of the polymer may be comprised of a spacer region. And the spacer region may be connected to the branched region via a first functional group. Such first functional group may be without limitation —NH₂, —OH, —PH₃, —COOH, —CHO, or —SH. Still further, the spacer region may comprise a linker region covalently bound to the first functional group.
In the substrate described above, the linker region may comprise a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group. Still further, spacer region may comprise a second functional group. The second functional group may include without limitation —NH₂, —OH, —PH₃, —COOH, —CHO, or —SH. The second functional group may be positioned at the terminus of the linear region and a protecting group may be bound to the terminus of the linear region. The protecting group may be acid labile or base labile.
The surface materials on which dendron thin film can be introduced are disclosed in US Publication No. 20050037413A1, the entire content of which is incorporated hereinto by reference. Such materials are used in the present invention.
In yet another embodiment of the invention, the substrate described above may consist of semiconductor, synthetic organic metal, synthetic semiconductor, metal, alloy, plastic, silicon, silicate, glass, or ceramic. In particular, the substrate may be, without limitation, a slide, particle, bead, micro-well plate, AFM (atomic force measurement) cantilever or porous material. The porous material may be a membrane, gelatin or hydrogel. And particularly, the bead may be a controlled pore bead.
It has been known that an organic thin film introduced on a glass surface by using the silane reaction is not stable in a buffer solution at a high temperature. (Anal. Chem. 2004, 76, 1778-1787). Even though its unstability in a buffer solution at a high temperature was confirmed again in this experiment, the present inventors found that the thermal stability of the organic thin film was dependent on the type of organic thin film.
Thus, the substrate is modified with the dendron. That is, the dendron-modified surface of the substrate is obtained by chemically modifying with the dendron, after treating the substrate with a silane compound having a hydroxyl group. Examples of silane compounds include (3-glycidoxypropyl) methyldiethoxysilane (GPDES) and N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (TPU), but they are not limited thereto.
Herein, the term “polynucleotide synthesis” includes polynucleotide synthesis, chain extension, and amplification. For example, the term includes DNA or RNA polymerizaton such as DNA or RNA synthesis, DNA or RNA chain extension, PCR, RT-PCR, random priming nucleic acid synthesis, or the other similar polynucleotide synthesis, chain extension, or any amplification method. The term, “polynucleotide” means DNA, RNA, oligonucleotide, cDNA, nucleotide analog or a combination thereof.
Any one of the enzymes used in standard PCR and RT-PCR, including Tag DNA polymerase and the modified Tag DNA polymerase, can be used for the present invention. In an embodiment of the present invention, the PCR method is performed by a denaturing step, an annealing step, and an extension step with Taq polymerase or a polymerase derived from Taq polymerase.
Herein, the term, “reaction buffer solution” is referred to a buffer solution used in amplification methods such as PCR, RT-PCR, random priming method, and the other similar amplification method. Examples of the buffer solution includes buffer solution 1 (50 mM KCl, 10 mM Tris-HCl, and 1.5 mM MgCl₂), buffer solution 2 (10 mM Tris-HCl, 40 mM KCl, 1.5 mM MgCl₂), and buffer solution 3 (50 mm Tris-HCl, 10 mM MgCl₂, 1 mM DTT, 50 μg/ml BSA), but are not limited thereto.
The present invention uses the method of immobilizing the biomolecules disclosed in US publication No. 20050037413A 1.
The present invention relates to a method for synthesis of polynucleotide on dendron-modified surface of substrate, where the dendron-modified surface is obtained by chemically modifying with a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface, and a terminus of the linear region of the dendron is functionalized.
The polynucleotide synthesis can be performed using the following three methods for example.
In an embodiment of the present invention for the synthesis of polynucleotide on the dendron-modified surface of substrate, the polynucleotide is synthesized by reacting (a) at least a primer immobilized on the dendron; with (b) a solution comprising polymerase, dNTP or NTP, and template DNA or RNA.
In another embodiment of the present invention for the synthesis of polynucleotide on the dendron-modified surface of substrate, the polynucleotide is synthesized by reacting (a) template DNA or RNA immobilized on the dendron; with (b) a solution comprising polymerase, dNTP or NTP, and primers.
In further embodiment of the present invention for the synthesis of polynucleotide on the dendron-modified surface of substrate, the polynucleotide is synthesized by reacting (a) polymerase immobilized on the dendron; with (b) a solution comprising dNTP or NTP, primers and template DNA or RNA.
In yet another embodiment of the present invention, the synthesis of polynucleotide can be performed under a high temperature, or under the thermal cycles that heating and cooling are repeated. The reaction temperature of polynucleotide synthesis can be different depending on the enzymes and synthesizing methods. For example, the reaction temperature of polynucleotide synthesis is 30° C. to 100° C., preferably 35° C. to 100° C, and more preferably 70° C. to 98° C.
In addition, the present invention provides a method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate under a thermal stress. The thermal stress can result from repetitive heating and cooling and can be long lasting. For example, the thermal stress can be a high temperature, such as a temperature in the range of 60 to 100□, and preferably 70 to 100□. The substrate is chemically modified with a dendron such that a plurality of termini of the branched region of the dendron are bound to the surface, and a terminus of the linear region of the dendron is functionalized. In the method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate, the substrate is chemically modified with the dendron, after treating the substrate with a silane compound having a hydroxyl group. The dendron, substrate, and biomolecules as described above can be used.
The following Example 1 shows that the dendron-modified surface had sufficient thermal stability in a reaction buffer solution. In the Example 2, the chain extension could be performed efficiently and successfully under a high temperature and also under a low temperature. It confirmed that the dendron-modified surface was suitable for PCR, RT-PCR, random priming and the other similar amplification methods.
The present invention is further explained in more detail with reference to the following examples. The scope of the present invention, however, is not limited to the following examples.

EXAMPLE 1

Preparation of Dendron-Modified Substrate

The two types of the modification (9-acid/GPDES substrate and 9-acid/TPU substrate) were employed for the substrate by using the two silane agents GPDES and TPU.

Example 1.1

Materials
The silane coupling reagents, (3-glycidoxypropyl)methyldiethoxysilane (GPDES) and N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (TPU) was purchased from Gelest Inc. and all other chemicals were of reagent grade from Sigma-Aldrich. Reaction solvents for the silylation are anhydrous ones in Sure/Seal bottles from Aldrich. All washing solvents for the substrates are of HPLC grade from Mallinckrodt Laboratory Chemicals. Glass slides (2.5×7.5 cm) were purchased from Corning Co. Ultrapure water (18 M Ω/cm) was obtained from a Milli-Q purification system (Millipore).

Example 1.2

Cleaning the Substrates
Glass slide as a substrate was immersed into Piranha solution (conc. H₂SO₄:30% H₂O₂=7:3 (v/v)) and a reaction bottle containing the solution and the substrates was sonicated for an hour. The plates were washed and rinsed thoroughly with a copious amount of deionized water after the sonication. The clean substrates were dried in a vacuum chamber (30-40 mTorr) for the steps to be followed.

Example 1.3

Preparing the Hydroxylated Substrates
The above clean substrates were soaked in 180 ml toluene solution with 1.0 ml (3-glycidoxypropyl)methyldiethoxysilane (GPDES) for 4 hours. After the self-assembly, the substrates were washed with toluene briefly, placed in an oven, and heated at 110° C. for 30 minutes. The plates were sonicated in toluene, toluene-ethanol (1:1 (v/v)), and ethanol in a sequential manner for 3 min at each washing step. The washed plates were dried in a vacuum chamber (30-40 mTorr). GPDES-modified substrates were soaked in a neat ethylene glycol (EG) solution at 80-100° C. for 8 h. After cooling, the substrates were sonicated in D.I water and ethanol in a sequential manner each for 3 min. The washed plates were dried in a vacuum chamber (30-40 mTorr).
Clean slide glass was immersed into anhydrous toluene (20 mL) containing N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (TPU) as a silane coupling agent (0.20 mL) under nitrogen atmosphere, and placed in the solution for 6 h. After silylation, the substrates were washed with toluene, baked for 30 min at 110° C. The substrates were immersed in toluene, toluene-ethanol (1:1 (v/v)), and ethanol in a sequential manner, and they were sonicated for 3 min in each washing solution. The substrates were rinsed thoroughly with toluene and methanol in a sequential manner. Finally the substrates were dried under vacuum (30-40 mTorr).

Example 1.4

Preparing the Dendron-Modified Substrates
The above hydroxylated substrates were immersed into a methylene chloride solution dissolving (9-anthrylmethyl N-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)methyl]amino}carbonyl)propylcarbamate) (or 9-acid) (0.5 mM) and a coupling agent, 1-[-3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) or 1,3-dicyclohexylcarbodiimide (DCC) (5 mM) in the presence of 4-dimethylaminopyridine (DMAP) (4 mM). After 3 days at room temperature, the plates were sonicated in methanol, water, and ethanol in the respective sequence each for 3 minutes. The washed plates were dried in a vacuum chamber (30-40 mTorr) for the step to be followed.

Example 1.5

Preparing the NHS-Modified Substrates
The dendron-modified substrates were immersed into a methylene chloride solution with 0.1 M trifluoroacetic acid (TFA). After 3 hours, they were again soaked in a methylene chloride solution with 1% (v/v) triethylamine(TEA) for 10 minutes. The plates were sonicated in methylene chloride and ethanol each for 3 minutes. After being dried in a vacuum chamber, the deprotected substrates were incubated in acetonitrile solution with di(N-succinimidyl)carbonate (DSC) (25 mM) and DIPEA (1.0 mM). After 4 hours of reaction under nitrogen atmosphere, the plates were placed in a stirred dimethylformamide solution for 30 min and then were washed briefly with methanol. The washed plates were dried in a vacuum chamber (30-40 mTorr) for the step to be followed.

EXAMPLE 2

Thermal Stability of the Immobilized DNAs on Dendron-Modified Surface

To test the thermal stability of the biomolecules on several different surfaces, Silanted slide, Dendron/EG/GPDES slide, and Dendron/TPU slide were used in this example. This example is to compare how DNA molecules immobilized on the dendron-modified surface of the present invention and on the aminosilane treated surface used in the conventional art were maintained stably in buffer solution at a high temperature. The Silanted slide (TeleChem International, Inc) which was treated with aminosilane was used as a comparative example.
Dendron/EG/GPDES slide, and Dendron/TPU slide were the same as those of Example 1.
The oligonucleotides used in this example included an amino group at 3′ end and Cy3 dye at 5′ end as follows:
5′Cy3-TTT TTT TTT T-NH₂-3′ (SEQ ID NO: 1)
A PCR buffer solution (50 mM KCl, 10 mM Tris-HCl, and 1.5 mM MgCl₂(pH 7.4)) was used as the buffer solution for measuring the thermal stability.
The oligonucleotides including a fluorescent dye were spotted on the dendron-modified surface of Example 1 with a microarrayer and the surface was incubated for a sufficient time to allow the oligonucleotides to be immobilized on the surface. Unreacted oligonucleotides were removed by rinsing with a washing buffer. The glass slide was dried, and then the fluorescence signal of the immobilized oligonucleotides was measured using a laser fluorescent scanner.
The glass slide showing the fluorescent signal was immersed in a PCR buffer solution at a temperature of 92-98□ for 5 minutes, washed with deionized water and dried before the fluorescence signal of the immobilized oligonuleotides was measured using a laser fluorescent scanner. Again, the glass slide showing the fluorescent signal was immersed in a PCR buffer solution at a temperature of 92-98□ for 5 minutes, washed with deionized water and dried before the fluorescence signal of the immobilized oligonuleotides was measured using a laser fluorescent scanner. The repetitive experiments as described above were carried out and then the intensity of fluorescent signal was analyzed for different repetition numbers.
It has been known that an organic thin film introduced on a glass surface by using the silane reaction is not stable in a buffer solution at a high temperature. (Anal. Chem. 2004, 76, 1778-1787). Even though the unstability was confirmed here again, the present inventors found that the thermal stability of the organic thin film depends on the type of organic thin film. The Silanated slide used as a comparative example showed steep decrease of the fluorescent intensity as the repetition number increased (FIG. 3A and FIG. 3B). However, although the dendron-modified surface showed the decreased intensity of the fluorescent signal, the amount of decrease was significantly smaller compared to that of Silanated slide (FIG. 4A and FIG. 4B).
In addition, in the preparation method of the dendron-modified solid surface, the solid surface treated with TPU (N-(3-(triethoxysilyl)propyl)-o-polyethylene oxide urethane) (FIG. 5A and FIG. 5B) was shown to be thermally more stable than those treated with GPDES ((3-glycidoxypropyl)methyldiethoxysilane) and ethylene glycol (FIG. 4A and FIG. 4B). It suggested that the TPU organic thin film block off the salts in buffer solution approaching the glass slide surface efficiently, and minimized the damage of Si—O bond.

EXAMPLE 3

Thermal Cycle Reaction With Enzymes on Dendron-Modified Surface

Based on the thermal stability test in Example 2, PCR, RT-PCR or the other similar thermal cycles could be performed on the dendron-modified surface by carrying out a general PCR and RT-PCR procedures on the surface.
As a comparative example, the Silanted slide treated with aminosilane (TeleChem International, Inc.) was used in this example.
Dendron/EG/GPDES slide, and Dendron/TPU slide were the same as those of Example 1.
The Tag DNA polymerase generally used in PCR was used in this example. The buffer solution for Tag DNA polymerase included 40 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl₂, but could be different according to the enzymes used. The buffer solution for DNA polymerase can be adjusted depending on the enzyme used.
The oligonucleotides used in this example included an amino group at 3′ end and Cy3 dye at 5′ end as follows:
5′-Cy3-ACA AGC ACA GTT AGG-NH₂-3′ (SEQ ID NO: 2)
The oligonucleotides including a fluorescent dye were spotted on the dendron-modified surface of Example 1 with a microarrayer and the surface was incubated in a sufficient time to allow the oligonucleotides to be immobilized on the surface. Unreacted oligonucleotides were removed by rinsing with a washing buffer. The glass slide was dried, and then the fluorescence signal of the immobilized oligonucleotides was measured using a laser fluorescent scanner.
The glass slide was immersed in a buffer solution containing 100 μM dATP, 100 μM dTTP, 100 μM dCTP, 100 μM dGTP, and Tag DNA polymerase, and heated at 94° C. for 2 minutes. Then, the heating cycle which was at 94° C. for 20 seconds, at 60° C. for 20 seconds, and at 72° C. for 20 seconds was repeated at 20 cycles sequentially, and then was at 72° C. for 7 minutes for the last step. The glass slide was washed with deionized water, dried, and then the fluorescence signal of the immobilized oligonucleotides was measured using a laser fluorescent scanner to compare the intensities of fluorescent signals in the samples obtained before and after PCR.
As a result, the comparative example of Silanated slide showed 20,000 of fluorescent intensity before PCR but showed a steep decrease of the intensity to 2,000 after PCR. On the other hand, dendron/TPU slide showed much smaller decrease of the fluorescent intensity from 15,000 before PCR to 11,000. This result was consistent with that of thermal stability obtained in Example 2, and represented that the dendron-modified surface provided organic thin film with more stability than the general silanated slide. Thus, the result confirmed that PCR, RT-PCR and other thermal cycle procedures similar to PCR or RT-PCR could be carried out efficiently.

EXAMPLE 4

4.1. DNA Extension by Enzymes on Dendron-Modified Surface

The PCR and RT-PCR methods include a denaturing step, an annealing step, and an extension step. This Example was performed to measure the efficiency of the extension step on the solid surface.

The dendron-modified surface used in this Example was the same as that of Example 1, and the oligonucleotides to be immobilized on the surface were as follows.

TABLE 1


NAME	SEQUENCE(5′ to 3′)	SEQ ID NO

Primer

1	5′-NH2-gatcaccagcggcatcgag - 3′	3

Primer 2	5′-NH2-gatcaccaccggcatcgag -3′	4

Primer 3	5′-NH2-cgatcaccaacggcatcgag -3′	5

Primer 4	5′-NH2-cgatcaccatcggcatcgag -3′	6

Primer 5	5′-NH2-atcacccgcggcatcga -3′	7

The oligonucleotide as described in Table 1 are markers to detect katG gene of Mycobacterium tuberculosis, and particularly mutated kat G gene in codon 315. Primer 1 is designed for detect wild type Mycobacterium tuberculosis, and Primers 2 to 5 are designed for detecting a mutant type. Primers 1 to 5 had NH2 group on their 5′-end. The template DNA which the oligonucleotides detect is isolated from Mycobacterium tuberculosis, the 315 codon-containing gene fragment of isolated whole gene is only amplified with PCR where the dCTP-Cy5 was added to be labeled. The template DNA has a length of about 200 base pairs. Klenow DNA polymerase I and Tag polymerase I were used at their optimal temperature of 37° C. and 72° C., respectively.
FIG. 6 is a schematic view showing the DNA extension reaction following the binding between the primer DNA and the template DNA. Firstly, the primers including a fluorescent dye were spotted on the dendron-modified surface with a microarrayer, and the surface was incubated in a sufficient time for the oligonucleotides to immobilize on the surface. Unreacted primers were removed by rinsing with a washing buffer. The glass slide was then dried. The obtained DNA microarray was hybridized the template DNA at a specific temperature for 2 hours, and the unhybridized template DNAs were removed by washing with a buffer solution, and then were dried.

4.2. DNA Extension on Dendron-Modified Surface by Using Klenow DNA Polymerase I

The obtained DNA microarray was incubated in Klenow DNA polymerase I reaction buffer solution at 37° C. for 10 minutes in order to sufficiently soak DNA microarray on the slide, and then incubated with addition of a buffer solution including Klenow DNA polymerase I, 100 μM dATP, 100 μM dTTP, 100 μM dGTP, 50 μM dCTP, and 50 μM dCTP-Cy3 at 37° C. for 30 minutes. The DNA microarray was washed with PBS, and dried before the fluorescent signal of DNA microarray was measured.
FIG. 7 is a fluorescent image of DNA microarray obtained from the chain extension by using Klenow DNA polymerase I: (A) Cy5 fluorescent image which showed the fluorescent signal of the Cy5-labeled Template DNA hybridized with primers, and (B) Cy3 fluorescent image which showed the fluorescent signal of the dCTP-Cy3 incorporated into the amplified DNA produced from DNA extension on the dendron-modified surface. The result of FIG. 7A represented that the hybridization between the primers and template DNA on the dendron-modified surface occurred with high selectivity. The result of FIG. 7B confirmed that the DNA extension on the solid surface by using Klenow DNA polymerase I was performed successfully. As shown in FIGS. 7A and 7B, the hybridization and polynucleotide synthesis performed by using Primer 1 which did not include a mismatched base, and Primer 2 to 5 which included 1 mismatched base pair showed that only Primer 1 provided complete hybridization with the template DNA and the DNA extension. Such result suggested that the dendron-modified surface of the present invention have high selectivity to matching and mismatching.

4.3. DNA Extension on Dendron-Modified Surface by Using Tag DNA Polymerase

The DNA microarray after DNA hybridization with template DNA was immersed in Tag DNA polymerase reaction buffer solution, and incubated at 72° C. for 10 minutes to allow the upper side of the DNA microarray on the slide to be wet sufficiently. The DNA microarray was immersed in a buffer solution containing Tag DNA polymerase, 100 μM dATP, 100 μM dTTP, 100 μM dGTP, 50 μM dCTP, and 50 μM dCTP-Cy3, and was incubated at 72° C. for 5 minutes. Then, the microarray was washed with PBS, and dried before its fluorescence was measured.
FIG. 8 is a fluorescent image of DNA microarray obtained from the chain extension by using Taq polymerase: (A) Cy5 fluorescent image which showed the fluorescent signal of the Cy5-labeled Template DNA hybridized with primers, and (B) Cy3 fluorescent image which showed the fluorescent signal of the dCTP-Cy3 incorporated into the amplified DNA produced from DNA extension on the dendron-modified surface.
The result of FIG. 8A represented that the hybridization between the primers and the template DNA on the dendron-modified surface occurred with high selectivity. The result of FIG. 8B confirmed that the DNA extension on the solid surface by using Taq polymerase was performed successfully.
As shown in FIG. 8A and FIG. 8B, in the case of the chain extension by using Primer 1 which does not include a mismatched base, and Primers 2 to 5 which include 1 bp mismatched, the hybridization and polynucleotide synthesis results showed that only Primer 1 completely hybridized template DNA, and thus provided the extension reaction. Thus, the dendron-modified surface showed high selectivity to the base pair matching and mismatching.
While the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that various modifications and substitutions can be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. A method for synthesis of a polynucleotide on a dendron-modified surface of a substrate, wherein the dendron-modified surface is obtained by chemically modifying with a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface and a terminus of the linear region of the dendron is functionalized.

2. The method according to claim 1, wherein the polynucleotide is synthesized by reacting (a) at least one primer immobilized on the dendron, with (b) a solution comprising polymerase, dNTP or NTP, and template DNA or RNA.

3. The method according to claim 1, wherein the polynucleotide is synthesized by reacting (a) template DNA or RNA immobilized on the dendron, with (b) a solution comprising polymerase, dNTP or NTP, and primers.

4. The method according to claim 1, wherein the polynucleotide is synthesized by reacting (a) polymerase immobilized on the dendron, with (b) a solution comprising dNTP or NTP, primers, and template DNA or RNA.

5. The method according to claim 1, wherein the dendrons are spaced at regular intervals between about 0.1 nm and about 100 nm among the linear functionalized groups.

6. The method according to claim 1, wherein the terminus of the branched region is functionalized with —COZ, —NHR, —OR′, or —PR″3, wherein Z is a leaving group, R is an alkyl, R′ is alkyl, aryl, or ether, and R″ is H, alkyl, or alkoxy.

7. The method according to claim 1, wherein the functional group is —NH2, —OH, PH3, —COOH, —CHO or —SH.

8. The method according to claim 1, wherein the linear region comprises a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group.

9. The method according to claim 1, wherein the substrate is glass, semiconductor, metal, plastics, silicone, silicate, metal alloy, or synthetic organic metal.

10. The method according to claim 1, wherein the substrate is in a form of a slide, a particle, a bead, a micro-well plate, or a porous material.

11. The method according to claim 1, wherein the polynucleotide is DNA, RNA, oligonucleotide, cDNA, nucleotide analog, or a combination thereof.

12. The method according to claim 1, wherein the dendron-modified surface is obtained by chemically modifying with the dendron after treating the substrate with a silane compound having a hydroxyl group.

13. The method according to claim 1, wherein the polynucleotide is synthesized by a Klenow DNA polymerase I.

14. The method according to claim 1, wherein the synthesis of polynucleotide is carried out by using RT-PCR or PCR method.

15. The method according to claim 14, wherein the PCR method include a denaturing step, an annealing step, and an extension step with Taq polymerase or a polymerase derived from Taq polymerase.

16. A method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate under a thermal stress, wherein the substrate is chemically modified with a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface and a terminus of the linear region of the dendron is functionalized.

17. The method of stably maintaining a polynucleotide according to claim 16, wherein the thermal stress is a temperature of 60 to 100□.

18. The method of stably maintaining a polynucleotide according to claim 16, wherein the thermal stress is long lasting.

19. The method of stably maintaining a polynucleotide according to claim 16, wherein the thermal stress is put by repetitive heating and cooling.

20. The method according to claim 16, wherein the substrate is chemically modified with the dendron, after treating the substrate with a silane compound having at least a hydroxyl group.