Description MODIFIED GLASS FIBERS WITH MONOLAYERS OF

AMINOCALIXARENE DERIVATIVES AND

IMINECALIXARENE DERIVATIVES, AND OLIGO-DNA

MODIFIED GLASS FIBERS BY FIXING OLIGO-DNA'S TO THE

MONOLAYER, AND THE PREPARATION METHOD OF GENOTYPING STRIPS USING THE OLIGO-DNA MODIFIED

GLASS FIBERS Technical Field

[1] The present invention relates to a method of preparing a surface-modified glass fiber by bonding an aminocalixarene derivative or an iminecalixarene derivative on the surface of a glass fiber to form a monolayer, a method of preparing a glass fiber where oligo-DNAs are immobilized by immobilizing oligo-DNAs having consecutive guanine bases on the glass fiber prepared according to said method, and a method of preparing a rapid genotyping kit using the glass fiber where various oligo-DNAs are immobilized prepared according to said method, thereby allowing genotyping of various genes.

[2]

Background Art

[3] Since a human genome map was completed in 2000 in a Human Genome Project conducted competitively by the Human Genome Research team and Celera Genomics, researches on the function of a gene and on the detection of a mutated gene have been done worldwide, and oligo DNA chips which is manufactured by using the results of the researches, where an oligomer DNA is bonded and immobilized and which is capable of genotyping various organisms, has been developed around the world.

[4]

[5] Particularly, since a oligo DNA chip can genotype viruses, many researches have been done around the world on an oligo DNA chip prepared by immobilizing several oligo DNAs on a substrate of an oligo DNA chip, such as a DNA chip for acurrately analyzing the route of a virus infection, confirming whether the virus is harmful or not, and predicting the possibility of cancer occurring by genotying the virus group which causes cancer.

[6]

[7] The DNA chip for genotyping HPV virus, which has been developed recently and

spotlighted, is being developed as a DNA chip which can genotype donzens of types of human papiloma viruses (HPV). It is reported that in the case where a virus of a specific genotype is present, the chances of cervical cancer occurring in the future is higher than 90%. Accordingly, said chip has been recognized to be important as a bio chip for prevention of diseases which can predict the chances of a disease occurring, and as a result, has been granted an official permit by the Korea Food and Drug Administration for the first time in the world.

[8]

[9] However, DNA chips prepared from modified glass slide where various DNAs are immobilized by using various physical and/or chemical methods go through user handling processes, such as loading, hybridization, washing, etc. In processing many samples by using human hands, there may be following problems, which need to be dealt with surely.

[10]

[11] 1) Human hands may not operate uniformly. In this regard, even a small difference in washing time or in the speed of dispensing an aqueous solution for washing, etc. may lead to a considerable error. Accordingly, the test results may change depending on the level of the person handling a DNA chip.

[12] 2) In a hybridization reaction conducted at a high temperature of about 50 ⁰ C for a long time of from about 30 minutes to about 2 hours, as a solvent evaporates, the number of bonding in the solution, e.g., the concentration of DNAs which could particpate in the hybridization, increases sharply. In such case, the resulting signals may differ by several times, which makes difficult a positive/negative determination.

[13] 3) Particularly, there are cases where fluorescence is visible at a position where no fluorescence should be detected, thereby misleading to conclude that the corresponding DNA is present; that is, there are cases where nonspecific DNA expression occurs, which makes difficult precise genotyping.

[14] 4) In the case of a DNA chip, experiments on 4 to 8 samples are conducted in one

DNA chip, and accordingly, there are often the cases where several coated solutions are mixed together, causing a serious problem that a sample which should be read negative reads positive. Accordingly, it is necessary to develop new genotyping technology where individual samples can be loaded and analyzed separately.

[15] 5) Particularly, a DNA chip based on a glass slide is difficult to manufacture in an easy to use form. Accordingly, it is necessary to develop technology which can produce an article maintaining a performance as a DNA chip and being manufactured in an easy to use form from a material having good processability.

[16] 6) In addition, one of the biggest factors preventing the wide use of a DNA chip is that, in order to confirm the final anaylsis result of the chip, an expensive scanner

should be used. Most of the current scanners amount to tens of thousands of dollars, which has hindered the development and use of a DNA chip allowing genotyping of various viruses. In order to solve this problem, it is necessary to develop a genotyping strip which can carry out genotyping by using an analyzer of a low price between hundreds and thousands of dollars, and technology of modifying a glass fiber which is essential to manufacture such a strip, that is, a glass fiber which can immobilize DNAs and can be easily processed, and technology of immobilizing various DNAs on a modified glass fiber to allow analysis of various genes, that is, genotyping of various genes.

[17]

Disclosure of Invention Technical Problem

[18] In order to solve the problem of the conventional DNA chips prepared by immobilizing oligo-DNAs on a glass slide, the present invention adopts the following methods. The research team of the present invention has developed technology of preparing a glass slide where oligo-DNAs having 7-15 consecutive guanine bases are spontaneously immobilized. Such a glass slide is prepared by applying an aminocalixarene derivative of formula 1 and an iminecalixarene derivative of formula 2 to an amine-modified glass slide to form an aminocalixarene monolayer and an iminecalixarene monolayer, respectively. In case of coating oligo-DNAs in the form of a solution on this glass slide, various kinds of oligo-DNAs are spontaneously immobilized. Accordingly, this glass slide is used to prepare a DNA chip easily and with high reproducibility.

[19]

[20] In order to immobilize various DNAs on a glass fiber, the present invention uses the fact that the surface of a glass fiber is the same as that of a glass slide. The present invention modifies the surface of a glass fiber with amine according to the known method disclosed in Langmuir, 1996, VoI 12, pp 5338-5342. Then, the present invention applies an aminocalixarene derivative of formula 1 and an iminecalixarene derivative of formula 2 to an amine-modified glass fiber to form an aminocalixarene monolayer and an iminecalixarene monolayer, respectively. Then, a surface of a glass fiber is coated with a solution including DNAs in the same way as for a glass slide to prepare a glass fiber where DNA is immobilized. That is, the present invention is aimed at establishing technology of preparing a glass fiber where several DNAs are immobilized, and a method of preparaing a genotyping strip using the glass fiber. Particularly, the present invention provides technology of preparing a genotyping strip, which identifies a genotype by bonding one or more of genes or a product obtained by

subjecting the solution to a polymerase chain reaction (PCR) in a solution with a probe DNA bonded onto a glass fiber by hybridization.

[21]

Technical Solution

[22] The present invention provides a method for the preparation of a surface-modified glass fiber, where said glass fiber is prepared by bonding an aminocalixarene derivative of formula 1 and an iminecalixarene derivative of formula 2 onto the surface of an amine-modified glass fiber according to the method of Figs.1 and 2, to form a monolayer.

[23]

[26] where, R , R , R , R , R , R , R , R R' , R' , R' , R' , R ¹ , R' , R' and R' are inde-

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 pendently selected from the group consisting of -H, -CH , -C H , -C H , -OCH , -Cl, -C H , -OH, -OCH CH , -Br, -CF , -OCH C H -OC H , -OC H CH , -OC H C(CH ) , -

6 5 2 3 3 2 6 5, 6 5 6 4 3 6 4 3 3

OC 6H4CF3, -OC6H4Cl, -OCOCH 3, -NHCOCH 3, -CONHCH 3, -CN, COOH, and -COOR where R represents -CH or -C H ; Y , Y , Y and Y are independently selected from the group consisting of -H, -(CH ) -CH=O, -(CH ) -SH, -(CH CH O) -CH CH -

2 n 2 n 2 2 m 2 2

CH=O, -(CH CH O) -CH CH -SH, -(CH ) -C H -(CH ) -Z and -CO-(CH ) -CH -

2 2 m 2 2 2 m 6 4 2 c 2 m-1 6 4

(CH ) -Z, where, n= 2-15, m= 1-10, c= 0-10, Z is a group selected from the group consisting of -SH, -CHO, -COOH and -NH , and -C H - and -C H are defined as

2 6 4 6 5 phenyl group.

[27] [28] [formula 2]

[29]

[30] where, R , R , R and R are independently selected from the group consisting of -H, -CH , -C H , -C H , -OCH , -Cl, -C H , -OH, -OCH CH , -Br, -CF , -OCH C H -OC

3 2 5 3 7 3 6 5 2 3 3 2 6 5, 6

H , -OC H CH , -OC H C(CH ) , -OC H CF , -OC H Cl, -OCOCH , -NHCOCH , - CONHCH , -CN, COOH, and -COOR where R represents -CH or -C H ; Y , Y , Y

3 3 2 5 1 2 3 and Y are independently selected from the group consisting of -H, -(CH ) -CH=O, - (CH ) -SH, -(CH CH O) -CH CH -CH=O, -(CH CH O) -CH CH -SH, -(CH ) -C H -

2 n 2 2 m 2 2 2 2 m 2 2 2 m 6 4

(CH where, n= 2-15, m= 1-10, c= 0-10, Z is a group selected from the group consisting of -SH, -CHO, -COOH and -NH , and -C H

2 6

4 - and -C 6 H 5 are defined as r pheny ^J l g orou rp.

[31] [32] The aminocalixarene derivative of formula 1 and the iminecalixarene derivative of formula 2 may be prepared according to the method disclosed in Korean Patent Application Nos. 10-2005-0096322, 10-2005-0103857, 10-2005-0105340 and 10-2005-0110824, which are incorporated herein by reference.

[33] [34] With regard to the method for forming a monolayer on a glass fiber, as described in the explanation of Fig. 3, a glass fiber has a thickness from about 10 nm to several hundred μm, and a glass fiber whose surface is abundant with a silnaol (-SiOH) function group is modified to be a glass fiber having an amine terminal by a chemical reaction (Refer to Langmuir, 1997, VoI 13, pp 4305-4308; Langmuir, 1996, VoI 12, pp 5338-5342). Then, the glass fiber having an amine terminal may be immersed in a solution where the compound of formula 1 or 2 is dissolved in a concentration of 0.1-5.0 mM in an organic solvent such as CHCl , etc, for about 1 to about 24 hours. Then, the glass fiber may be washed with the same solvent and dried to obtain a

surface-modified glass fiber where a monolayer of aminocalixarene or aminecalixarene is formed.

[35]

[36] By using said method, the present invention provides a glass fiber having a self- assembled monolayer, where an aminocalixarene derivative of formula 1 or an iminecalixarene derivative of formula 2 is attached to a surface of a glass fiber by a chemical bonding such as an imine bonding, an amine bonding obtained by reducing imine bonding, a thiol bonding, an ester bonding, an ether bonding, an amide bonding, etc.

[37]

[38] In the method of immobilizing DNA onto a modified glass fiber illustrated in Figs. 4 and 5, oligo-DNAs having consecutive guanine bases may be spontaneously immobilized in an immobilization solution where 60-600 mM of ions are present to prepare a glass fiber where DNA is immobilized. The method of immobilizing DNA according to the present invention includes: preparing an immobilization solution by dissolving an oligo-DNA having consecutive guanine bases (e.g. 9 consecutive guanine bases) in a BMT dispensing solution (solution of 600 mM of ammonium ions); dispensing said immobilization solution to said glass fiber modified with a monolayer of aminocalixarene or aminecalixarene; and immobilizing the oligo-DNA. Said method may optionally include washing said glass fiber, and blocking the locations except those where oligo-DNA is immobilized. In said method, the immobilization step may be conducted preferably at the room temperature for about 1 to about 4 hours. The blocking step may include putting the washed glass fiber into a blocking solution (lx~4xSSC, 0.1-5.0% caseine) and treating it for 10~30 minuntes.

[39]

[40] When compared according to the method of Fig. 9., it was observed that the concentration of the DNAs immobilized onto the thus-prepared glass fiber is about 1/2 to 1/3 times of the theoretical maximum immobilization amount, which shows that the present invention achived technology of preparing a glass fiber capable of immobilizing DNA in an amount near the theoretical maximum. Fig. 5 and Fig. 6 show the hybridization results of various genes according to the degree of consistency of the bases. In a narrow space of about 1.4 cm, DNAs were immobilized into 8 straight lines, and then each line was coated with a fluorescence-labeled gene to conduct hy- bridzation. A glass fiber may firmly immobilized DNAs in an amount such that the fluorescence is individually identified at the immobilized location by hybridization, to maintain a spaced state and its immobilized location while a solution developes, which is shown in the experiment result in Fig. 5. Example 3 shows a method of immobilizing DNAs, and Example 4 shows a process of coating fluorescence-labled

genes.

[41]

[42] In addition, the present invention provides a method for genotyping capable of identifying a gene having a specific base sequence, including:

[43] - developing a solution including fluorescence-labeled genes having an amplified nucleic acid, over the glass fiber according to the present invention; and

[44] - bonding genes having a specific base sequence, e.g., a specific genotype of virus, with a complementary probe oligo-DNAs immobilized on the surface of the glass fiber by hybridization.

[45] Fig. 6 shows a result in genotyping, i.e., the hybridization result according to the consistency of genes. In order to obtiain the hybridization results, several genes of different genotypes are immobilized and a fluorescence-labeled gene of one genotype is coated according to Example 5. Actual experimental results and analysis of the results are presented in Fig. 6. Accroding to the results, the location where the genotypes do not match shows little fluorescene intensity, but the location where the genotypes match shows 10 to 40 times higher fluorescence intensity.

[46]

[47] The genotyping method according to the present invention may be carried out at a low temperature from about 4 ⁰ C to about 2O ⁰ C, at a room temperature from about 20 ⁰ C to about 3O ⁰ C, or at a high temperature from about 30 ⁰ C to about 6O ⁰ C. Meanwhile, since the polymerase chain reaction was disclosed in 1971 by K. Kleppe and G, Khorana in the Journal of Molecular Biology , vol. 56, pp 341-361, the hybridization of amplified gene has been conventionally conducted at a high temperature from about 40 ⁰ C to about 6O ⁰ C to increase selectivity. However, a result of the reaction carried out at a normal temperature as in the present application has not been published. Generally, the temperature of a lab is maintained at about 22°C to 25 ⁰ C, and hybridization may be carried out at a room temperature including said temperature. In the case of a hybridization reaction that can be carried out only at a high temperature (from about 3O ⁰ C to about 6O ⁰ C), hybridization can be carried out only when the temperature is raised by using an oven, etc. In the case of a hybridization reaction carried out at a low temperature (from about 4 ⁰ C to about 2O ⁰ C), hybridization is carried out while using a device for lowering the temperature, such as a chiller. Hybridization may be categorized according to the reaction temperature and a device for maintaining the temperature.

[48]

[49] In the present invention, a glass fiber is modified with macromolecules such that probe DNAs having consecutive guanine bases can be immobilized on the glass fiber. In addition, in the present invention, the probe oligo-DNAs are appropriately spaced

from each other by the consecutive guanine bases, therey making it possible to carry out hybridization even at a room temperature or a low temperature. Different from general expectation, the probe DNA immobilized on the modified glass fiber is hybridized at a room temperature or a low temperature, and obtains fluorescence intensity showing that the same or higer hybridization rate than in the hybridization at a high temperature has been illustrate. Fig. 6 and Fig. 9 illustrate a fluorescence data obtained from hybridization at a room temperaute, which can be easily used, among various fluorescence data.

[50]

[51] In addition, compared with the data obtained from a DNA chip based on conventional glass slide, the present invention provides a modified glass fiber having remarkably excellent selectivity and reduced nonspecificity, and techonology of preparing and using a strip for rapid genotyping (e.g., virus genotying strip) by using said glass fiber. A genotyping strip may include a glass fiber including at least one type of oligo-DNA having consecutive guanine bases, and may have a sample inlet on a part of the glass fiber on which oligo-DNA is not immobilized. The genotyping strip may be prepared in the form of a rapid strip or a genotyping kit including the strip.

[52]

[53] In addition, the present invention provides a method for genotyping capable of identifying a gene having a specific base sequence, including:

[54] - injecting a solution including fluorescence-labeled genes having an amplified nucleic acid, through the sample inlet of the strip for genotyping according to the present invention;

[55] - developing the solution over the glass fiber; and

[56] - bonding genes having a specific base sequence with a complementary probe oligo-

DNAs immobilized on the surface of the glass fiber by hybridization.

[57]

[58] In addition, the present invention provides a genotyping method including: coating a glass fiber with a solution including genes (RNA, DNA, etc.) having specific sequences; bonding the genes having a specific base sequence with a complementary oligo-DNAs having consecutive guanine bases immobilized on the surface of the glass fiber by hybridization; and measuring the total amount of the bonded genes by spectroscopic methods using fluorescence, or visible rays, etc.

[59]

Advantageous Effects

[60] To sum up, the present invention provides a glass fiber modified with macro- molecules, where oligo-DNAs are immobilized, the preparation method thereof, and

the method of preparing a genotyping strip using said glass fiber where oligo-DNAs are immobilized. In addition, the present invention achieved technology of preparing and using a surface-modified glass fiber where oligo-DNAs are immobilized. The present invention can solve the problems that a conventional DNA chip cannot be widely used due to the use of an expensive scanner, and the test results change depending on the person who uses the chip, and the chip is not easy to handle. In addition, the present invention achieved technology of preparing a genotyping strip which is easy to use and requires little processing, etc, thereby can remarkably improve problems occurring with a DNA chip based on a conventional glass slide and suffered by many users. In addition, a glass fiber has a characteristic that a solution developes thereon at a specific speed, as in NC membrane used for a rapid kit. Accordingly, when oligo-DNA is immobilized on a surface of a glass fiber, gene included in a solution developing at a specific speed is hybridized while developing through the uniformly immobilized DNAs at a constant speed. Accordingly, the present invention can prepare an article in the form of a strip, which does not need to be handled by a user except when injecting a sample in the form of a solution. As such, the present invention provides a chip that can be used without a user handling, thereby can remarkably increase the reproductibility of test results and increase convenience. In order to achieve the above effects, the present inventors have developed technology of modifying the surface of a glass fiber by immobilizing the oligo-DNA.

[61]

[62] Thus, the present invention provides world's first technology of preparing glass fiber by improving conventional technology of modifying a glass slide, thereby making it possible to prepare a genotyping strip where oligo-DNA is immobilized with a high density such that it can be used without user handling. In addition, the present invention achieved technology of preparing a genotyping strip using a glass fiber where oligo-DNA is immobilized. Not only the strip prepared according to this technology can solve the problem occurring when preparing and using conventional oligo-DNA chips, such as that the test results are not reproducible and that the test results change depending on the user, but also can provide technology of preparing a DNA chip in the form of a glass fiber-based strip, thereby allowing genotyping using an inexpensive analyzer (i.e., without using an expensive analyzer costing tens of thousands of dollars). The present invention can solve most of the problems occurring when preparing and using a DNA chip based on a conventional glass slide. Test results of the thus-prepared strip can be obtained by using a scanner of a low price from about hundreds dollars to thousands of dollars equipped with a linear motor. Fig. 5 shows a graph of actual experimental results obtained by a low-price scanner. The present invention is important in that it can solve the problem occurring when using con-

ventional DNA chips to allow various diagonosis using genotyping. [63]

Brief Description of the Drawings

[64] Figs. 1 and Fig. 2 are a diagram of the glass fiber whose surface is modified with amine according to the method of a paper published in Langmuir, 1996, VoI 12, pp 5338-5342, and a diagram showing the process of preparing a monolayer of macro- molecules by bonding the derivative of formula 1 or formula 2 with the amine- modified glass fiber.

[65]

[66] Fig. 3 is a picture of the glass fiber actually used for said preparation (Fusion 5

(Whatman Co., U.K.)), its enlargement, and its diagram. The glass fibers used in the present invention, including a product by Whatman Co. of U.K., Water Co. of U.S.A. and Millipore Co. of U.S.A. etc., are obtained by drawing a fine glass fiber with a diameter of 10 nm to several hundred μm from raw glass materials, and then weaving it into a membrane. Accordingly, said fiber may have a characteristic that a solvent applied on one part of the fiber developes by the force of gravity, the capillary action, microfludics, or by a combination of at least two of thereof. The present invention uses such characteristic of said glass fiber.

[67]

[68] Fig. 4 is a diagram showing the process of preparing glass fibers where oligo-DNA is immobilized by dispensing a solution where oligo-DNA having consecutive guanine bases is dissolved in the form of a line with a specific width on a glass fiber modified with a monolayer of macromolecules, by using a dispenser, etc.

[69]

[70] Fig. 5 shows the result of an actual experiment. In this experiment, when a solution including a fluorescence-labeled amplified gene obtained by a polymerase chain reaction (PCR) is applied on a glass fiber where oligo-DNAs having consecutive guanine bases are coated in the form of lines, and as a result, where multiple oligo- DNAs are immobilized in the form of multiple lines, the solution developes by itself, and then genes of which genotypes are complementary to the immobilized probe oligo- DNAs are bonded by hybridization. This experiment used fluorescence-labeled genes in order to identify the bonded genes by measuring fluorescence intensity.

[71]

[72] Fig. 6 shows actual experiment results obtained by letting a solution including fluorescence-labeled genes flow on a glass fiber where multiple oligo-DNAs are coated in the form of lines to be immobilized, and conducting an analysis by using a fluorescence scanner. Strong fluorescence is detected at locations where the base

sequences of the genes are identical with base sequences desired to be detected, and no fluorescence is detected at locations where the base sequences are not identical with base sequences desired to be detected. Thus, in this experiment, fluorescence-labeled genes are detected only at locations where the base sequences are identical.

[73]

[74] Fig. 7 is a diagram of a strip prepared by placing a glass fiber where multiple oligo-

DNAs are immobilized into a strip and then covering it with a lid. When a solution including amplified genes obtained by PCR was injected through a sample inlet of the strip, gene hybridization results shown in Figs. 5 and 6 were obtained. The results were analyzed mainly by spectrometry.

[75]

[76] Fig. 8 is a diagram showing the method of analyzing the results by spectrometry after the process of Fig. 7 where the solution developes. Fig. 8 shows the process of placing a glass fiber where multiple oligo-DNAs are immobilized in the form of lines into a strip and then injecting a solution including varous genes through a sample inlet as in Fig. 7 and then, after a certain period, observing the amount of genes bonded to each of the lines with the naked eyes or by using an optical instrument, and then representing it as a magnitude of a signal. Genotypes of gene(s) present in the solution can be identified by oberving the difference in the magnitude. Actual experiment results obtained by detecting fluorescence are presented in Fig. 6. From Fig. 6, it can be confirmed that considerable levels of fluorescence is detected only when the genotypes are identical.

[77]

[78] Fig. 9 shows the results of actual experiments comparing the fluorescence intensity obtained when the theoretical maximum amounts of fluorescence-labeled genes that can be hybridized are coated and dried, with the fluorescence intensity obtained by hybridization with actually immobilized DNAs. Here, the theoretical maximum amount of fluorescence-labeled genes that can be hybridized may be obtained by calculating the maximum amount of oligo-DNAs having consecutive guanine bases which can be immobilized on the surface of a glass fiber when immobilizing the DNAs in the form of lines, and then calculating a maximum amount of fluorescence-labeled genes that can be hybridized with them. Here, this calcultion may be made on the assumption that fluorescence-labeled genes are hybridized with the immobilized oligo-DNAs at 1 : 1 ratio (In order to confirm a decrease of fluorescence intensity from theoretical maximum amount i.e., xl, fluorescence intensity may be measured using decreased concentrations genes such as xl/3, xl/9, etc.). Meanwhile, the results of the experiment of hybridizing fluorescence-labeled genes with actually immobilized DNAs have been confirmed repetedly by using 5 lines. The fluorescence intensity obtained from the

experiment was about 1/3 times the theorectial maximum fluorescence intensity, which shows that the present invention achieves excellent gene immobilization and hy- bridzation results.

[79]

[80] Fig. 10 is a picture showing a type of an assembled strip having a sample inlet, as used in Fig. 7 and Fig. 8.

[81]

[82] Fig. 11 to Fig. 16 show the experiment result obtained by assembling a modified glass fiber where probe oligo-DNAs of different genotypes are immobilized into a strip as shown in Fig. 10, and then letting fluorescence-labeled amplified genes develop on the strip, and then reading the result by using a fluorescence scanner as described in Example 7. Fig. 11 to Fig. 14 show the results obtained when applying amplified genes complementary to one probe genotype, and Fig. 15 and Fig. 16 show the actual results obtained by applying amplified genes complementary to at least two probe genotypes and then carrying out the same method. All the results show that in genotyping using a modified glass fiber, genes are hybridized selectively, and nonspefic hybridization does not occur.

[83]

Mode for the Invention

[84] The present invention will be described in further detail with reference to Examples.

However, it should be understood that the present invention is by no means restricted to such specific Examples.

[85]

[86] Example 1

[87] Method of forming a monolayer on an amine-modified glass fiber by applying a solution where 5,ll,17,23-tetradibenzylaminocalix[4]arene-l,3-hexanealdehyd e (TDBACAHA) is dissolved

[89] where X is the same as the functional group representing the connecting parts having aldehyde terminal groups of formula 1.

[90] TDBACAHA [91] 0.1-5.0 mM of 5,1 l,17,23-tetradibenzylaminocalix[4]arene-l,3-hexanealdehyde (TDBACAHA) among the derivatives of formula 1 was dissolved in an organic solvent such as CHCl etc. to prepare a solution. As shown in Fig. 1 and Fig. 2, a glass fiber where amine functional groups are attached (e.g., amine glass fiber substrate) was immersed in the thus-prepared solution for 1-24 hours, and then the glass fiber was sequentially washed with chloroform, acetone, and then finally with water, and then dried to form the aminocalixarene monolayer. Another aminocalixarene derivative monolayer was prepared according to the same method.

[92] [93] Example 2 [94] Method of modifying an amine-modified glass fiber by applying a solution where 5,H,17,23-tetrabenzyliminealkoxycalix[4]arene (TBICOCA) is dissolved (prepared according to conventional methods)

[95]

[96] TBICOCA [97] 0.1-5.0 mM of 5,l l,17,23-tetrabenzyliminealkoxycalix[4]arene (TBICOCA) among the derivatives of formula 2 was dissolved in an organic solvent such as CHCl etc. to prepare a solution. As shown in Fig. 3, a glass fiber where amine functional groups are attached (e.g., amineglass fiber substrate) was immersed in the thus-prepared solution for 1-24 hours, and then the glass fiber was sequentially washed with chloroform, acetone, and then finally with water, and then dried to form the iminecalixarene monolayer of the present invention. Another iminecalixarene derivative monolayer was prepared according to the same method.

[98] [99] Example 3 [100] Method of immobilizing oligo-DNAs by using a modified glass fiber to genotype various types of genes

[101] [102] A Biodot dispenser (Model No. XYZ 3050, U.S.A) was used for the oligo-DNA immobilization shown in Fig. 5. In order to genotype various types of genes, probe DNAs having completely complementary base sequence as listed in Table 1 was immobilized as in Fig. 5. That is, 3-30 pmol/μl of oligo-DNAs having consecutive 9 guanine bases were dissolved in a BMT dispensing solution (600 mM ammonium ion solution) to prepare 8 sets of an immobilization solution (600 mM ammonium ion solution; product name: BMT spotting solution-9G, manufactured by Biomatrix Technology Co. of Republic of South Korea). Said immobilization solution was dispensed at the rate of

5-50 mm/sec by using a dispensing nozzle. On a modified glass substrate (glass fiber), an aminocalixarene derivative monolayer or an iminecalixarene derivative monolayer as shown in Fig. 4 was formed according to the methods of Examples 1 and 2. Then, oligo-DNAs were immobilized in the form of lines of 0.5-5 mm in width on the thus- prepared glass substrate. After carrying out immobilization for 1-4 hours at the room temperature, the glass fiber was washed, and then immersed into 250 ml of BMT blocking solution (lx~4xSSC, 0.1-5.0% caseine; product name: BMT Blocking solution-9G, Biomatrix Technology Co., Republic of South Korea) for 10~30 minutes in order to block the locations where oligo-DNAs are not immobilized.

[103] Table 1 [Table 1] [Table ]

[104] [105] Example 4 [106] Method of detecting gene by using hybridization with a fluorescence-labeled target DNA

[107] [108] In order to carry out hybridzation with a fluorescence-labeled target DNA, 5 μl of fluorescence -labeled target DNA listed in Table 1 and 75 μl of BMT hyb-mixA (6xSSC, 20% formamide, 0.05% triton X-100; product name: BMT Hyb-solution-9G, Biomatrix Technology Co., Republic of South Korea) were put into a 1.5 ml tube to prepare 80 μl of a mixed solution. Then, the thus-prepared solution was injected through the sample inlet of an assembled strip. Then, hybridzation was carried out at a room temperature (20+5 ⁰ C) for 5-50 minutes. After hybridzation has been completed, 100-300 μl of 0. lx~4x SSC solution was injected and then the strip was separated. Then, the glass fiber was attached to a slide and then the fluorescence intensity was analyzed quantitatively by using a microarrayer scanner (GSI Lumonics, U.S.A.) or a linear scanner. Fig. 5 illustrates actual experiment results obtained by using a linear scanner. These results show that a glass fiber where oligo-DNAs are immobilized has been prepared, and that when applying various types of genes on a surface-modified

glass fiber and then letting a solution including a fluorescence-labeled target DNA flow over the glass fiber, genes in the solution are hybridized with various immobilized oligo-DNAs, and thereby the glass fiber can identify whether a specific type of gene is included in the solution.

[109] [110] Example 5 [111] Experiment of detecting a specific type of gene by using a surface-modified glass fiber

[112] In the oligo-DNA immobilization shown in Fig. 6, a Biodot dispenser (Model No. XYZ 3050, U.S.A.) was used. In order to determine nonspecific fluorescence, the probe oligo-DNAs having a completely complementary base sequence and the probe oligo-DNAs having a non-complementary base sequence as listed in Table 2 were immobilized as shown in Fig. 6 according to the method of Fig. 4. The same composition of an immobilization solution and the same dispensing method as those of Example 3 were used to prepare a glass fiber where DNAs are immobilized.

[113] Table 2 [Table 2] [Table ]

[114] [115] In order to apply a fluorescence-labeled primer or a PCR product and identify whether it is hybridized with the DNA immobilized on a glass fiber, the glass fiber substrate prepared above was assembled into a strip of the shape shown in Fig. 10. In order to carry out hybridization, 5 μl of fluorescence-labeled target DNA and 75 μl of BMT hyb-mixA were put into a 1.5 ml tube to prepare 80 μl of a mixed solution. Then, the thus-prepared solution was injected through the sample inlet of an assembled strip. Then, hybridzation was carried out at a room temperature (20±5°C) for 5-50 minutes. After hybridzation has been completed, 100~300 μl of 0.1x~4x SSC solution was injected and then the strip was separated. Then, the glass fiber was attached to a slide, and then the fluorescence intensity was analyzed quantitatively by using a mi-

croarrayer scanner (GSI Lumonics, U.S.A.). Fig. 6 shows actual experiment results. The results show that fluorescence is visible only at the line having a desired base sequence, and that when letting genes flow over a glass fiber by using capillary action, microfludics, or lateral flow, etc., a target DNA having a complementary base sequence can be specifically detected.

[116]

[117] Example 6

[118] Experiment of comparing with a theoretical maximum amount of genes that can be immobilized on the surface of a glass fiber

[119]

[120] In order to carry out an experiment of comparing with a theoretical maximum amount of DNAs that can be immobilized on the surface of a glass fiber, probe DNA having consecutive guanine bases and a complementary base sequence was prepared and applied in the form of 5 lines on a modified glass fiber according to the same method as that of Example 3. In order to apply a fluorescence-labeled primer or a PCR product and identify whether it is hybridized with the DNA immobilized on a glass fiber, the glass fiber substrate prepared above was assembled into a strip of the shape shown in the diagram of Fig. 7 or the picture of Fig. 10. Hybridization was carried out according to the same method as that of Example 4. After hybridzation has been completed, the glass fiber was detached from the strip. Then, the fluorescence intensity was analyzed quantitatively by using a microarrayer scanner (GSI Lumonics, U.S.A.) to obtain experimental data. In addition, in order to compare the experimental data with the theoretical maximum amount of genes that can be immobilized on the surface, a solution including a fluorescence-labeled gene in the concentration of Ix which is the same amount as the theoretical maximum amount of DNAs that can be immobilized on the surface was prepared. Then, said solution was diluted to 1/3, 1/9, 1/27, 1/81 folds. Then, each of the solutions was applied on a glass fiber in the form of a line by using a dispenser, and the glass fiber was dried. The results were read by using a fluorescence detector. Fig. 9 shows actual results.

[121]

[122] Example 7

[123] Method of detecting the lines emerging on the membrane of said modified glass fiber after probe DNAs are hybridized with amplified genes by measuring fluorescence

[124]

[125] A) Experiment of identifying the type of gene by using a product obtained by PCR by using a standard material

[126]

[127] Standard materials used in this Example were purchased from ATCC (American Type Culture Collection), and are as follows:

[128] Probe- 1 (ATCC 45150)

[129] Probe-2 (ATCC 45151)

[ 130] Probe-3 (ATCC 45152)

[131] Probe-4 (ATCC 45113)

[132] While using plasmid DNA among the above as a template and the primers in Table 3, PCR was carried out according to the following method. The primers used in the PCR was synthesized by Bioneer Co. Ltd. (Republic of South Korea) by our order. PCR was carried out by treating a reaction solution purchased from Bionia Co. Ltd., including 10 μl of PCR buffer, 1 μl of 1.5 mM MgCl , 250 uM dNTP, 30 mM KCl, 10 mM Tris- HCl(pH9.0), Taq polymerase (1 unit) and primer (10 pmol/μl), 7 μl of distilled water, and 1 μl of template DNA, at 94 ⁰ C for 5 minutes one time, and then 35 times repeating a treatment at 94 ⁰ C for 1 minute, at 45 ⁰ C for 45 seconds, and at 72 ⁰ C for 1 minute, and then treating the solution at 72°C for 5 minutes one time. Then, 5 μl of the thus- prepared reaction solution was applied to 2% agarose gel along with a DNA size standard maker, and then it was subjected to electrophoresis. Here, the electrophoresis gel was dyed by 0.00005% ethidium bromide solution. Whether the band emerging on each of the paths in the gel is valid was confirmed by using UV.

[133] Table 3 [Table 3] [Table ]

[134]

[135] B) Method of applying a fluorescence-labeled PCR product and hybridizing it with the probe DNA immobilized on a glass fiber and then conduting an analysis by observing fluorescence

[136]

[137] In the Probe DNA immobilization shown in Fig. 4, a Biodot dispenser (Model No. XYZ 3050, U.S.A.) was used. 7-30 pmol/μl of probe DNAs having 9 consecutive guanine bases, including probe 1, probe 2, probe 3, probe 4, HC (Hybridization control) listed in Table 4, were dissolved in a BMT dispensing solution (100 mM ammonium

ion solution) to prepare an immobilzation solution. Said immobilzation solution was dispensed at the rate of 20 mm/sec(0.7ul/Cm) by using a dispensing nozzle, and was applied on a modified glass fiber manufactured by Biomatrix Technology Co. The probe DNAs were applied in the form of a linear band of 0.2-3.0 mm in thickness and immobilized spontaneously. The immobilization was carried out for 1-24 hours at the room temperature. Then, in order to block the locations where oligo-DNAs are not immobilized, the glass fiber was washed, and then immersed into 250 ml of BMT blocking solution (4xSSC; including 1% caseine and 0.5% Poly Ethylene Glycol) for 10-30 minutes, and then was dried in a 40-50 ⁰ C incubator for 30 minutes to 4 hours to prepare the strip shown in Fig. 10.

[138] Table 4 [Table 4] [Table ]

[139] [140] C) Method of applying a fluorescence-labeled PCR product and hybridizing it with probe DNA immobilized on a glass fiber and then analyzing the materials by using fluorescence

[141] [142] After hybridizing a fluorescence-labeled primer or PCR product with the DNA immobilized on a glass fiber, in order to identify whether a specific gene is bonded, the glass fiber or strip prepared by immobilizing probe DNAs on the glass fiber substrate prepared in Example 2 according to the method of Example 7 -B) was assembled into a case of the shape shown in Fig. 10. Then, it was identified whether a specific gene is present by letting the PCR products developes over the glass fiber. In order to prepare a developing solution, 5 μl of PCR product prepared in Example 7-A) and 75 μl of BMT hyb-mix A were put into a 1.5 ml tube to prepare 80 μl of mixed solution. Then, the thus-prepared solution was heated in water at 100 ⁰ C for 3 minutes, and then cooled

on ice for 3 minutes. Then, the 80 μl of mixed solution was injected through the sample inlet of an assembled strip. Then, the strip was left at the room temperature (20~30°C) for 3-120 minutes so that the solution flows and hybridization can be carried out. Then, 100 μl of BMT Wa-B-2 (4xSSC) solution was injected, and after 5 minutes, the strip was separated. Then, the glass fiber (strip) was attached to a slide and then fluorescence intensity was analyzed quantitatively by using a microarrayer scanner (GSI Lumonics, U.S.A.). Actual results are shown in the strips at the top of Fig. 11 to Fig. 16. The graphs in the middle of Fig. 11 to Fig. 16 show analysis results obtained by using BMT HPV strip reader type-1, which is used for analyzing a strip in BMT. As can be clearly seen from the drawings, in the case of the samples including the amplified genes complementary to probe DNAs, specific hybridzation occurred at the bands (lines) where the complementary probe DNAs are immobilized, and hybridzation was carried out in an HC area, too, making it possible to identify whether the samples were dispensed. Meanwhile, no hybrization occurred at the location of probe DNA of different type. That is, the drawings show that a strip-type chip capable of genotyping a gene amplified by PCR with high selectivity and low nonspecific hybridization rate has been developed and experimented.

[143]

[144] The present invention discloses world's first technology of modifying the surface of a glass fiber such that DNA can be immobilized on it, said technology including a surface-modified glass fiber whose surface is modified by an aminocalixarene derivative of formula 1 or an iminecalixarene derivative of formula 2 to form a monolayer, a method of preparing the same, a modified glass fiber where DNA is immobilized, a method of preparing the same, a method of preparing a genotyping strip using the glass fiber where DNA is immobilized, etc. The present invention remarkably improved existing technology by solving the cost problem resulting from the use of expensive scanner, providing convenience by removing complex processing, and achieving reproductibility by virtue of the uniform developement of a solution.

[145]

[146] A glass fiber has a characteristic that a solution developes thereon at a specific speed, as in NC membrane used for a rapid kit. Accordingly, when oligo-DNA is immobilized on the surface of a glass fiber, gene included in a solution moving at a specific speed is hybridized at a constant speed while moving through the uniformly immobilized DNAs, thereby making it possible to prepare an article in the form of a strip which does not requre a user's handling except when loading a sample. The present invention can remarkably increase the reproductibility of test results and increase convenience. In order to achieve the above effects, the present inventors have developed technology of modifying the surface of a glass fiber such that that DNA can be immobilized on it.

Thus, the present invention achieved world's first technology of preparing a glass fiber where oligo-DNA is immobilized with a high density by improving conventional technology of modifying a glass slide. In addition, the present invention achieved technology of preparing a genotyping strip using the glass fiber where oligo-DNA is immobilized. Not only the strip prepared according to this technology can solve the problem occurring when preparing and using conventional oligo-DNA chips, such as that the test results are not reproducible and that the test results change depending on a user, but also can provide technology of preparing a DNA chip in the form of a glass slide-based strip, which allows genotyping using an inexpensive analyzer (i.e., without using an expensive analyzer costing tens of thousands of dollars). That is, the present invention is important in that it can solve the problem occurring when using conventional DNA chips, thereby allowing various diagonosis by using genotyping.