# Patent

## Title
Process of quality examining for microarray of biological material

## Abstract

The present invention relates a process of quality examining for microarray of biological material. More particularly, the present invention is directed to a process of quality examining for microarray of biological material, which comprises 1) a step for mixing probe and a compound which emits light or heat and does not react with said probe, 2) a step for microarraying the mixture obtained in step 1) on a substrate, and 3) a step for measuring light or heat emitted by the scanning of each spots of the microarray mixture.

## Background
TECHNICAL FIELD
The present invention relates a process of quality examining for microarray of biological material. More particularly, the present invention is directed to a process of quality examining for microarray of biological material, which comprises a step for mixing probe and a compound which emits light or heat and does not react with said probe, a step for microarraying the mixture obtained in step 1) on a substrate, and a step for measuring light or heat emitted by the scanning of each spots of the microarray mixture.
BACKGROUND ART
A microarray of biological material means a device for detecting genes or proteins wherein a large number of fragments of DNA, RNA or protein, are immobilized on a small substrate in high density. Such microarray chip is applied to researches for analyzing DNA mutations, RNA expressions and functions of proteins for understanding function of genes in large scale.
The microarray of a biological material (hereinafter, Biochip) may be classified into cDNA chip, oligonucleotide chip and protein chip on the basis of size of biological materials immobilized thereon. Full-length open leading frames or expressed sequence tag(EST)s having more than five (5) hundred base pairs can be immobilized on the cDNA chip. Oligonucleotides consisting of approximately 15 to 25 base pairs can be immobilized on the oligonucleotide chip.
The biochip on which plasmid DNA probe, oligonucleotide probe or protein is immobilized is primarily used for detecting gene expression, protein activity and DNA mutation. The most general applications of DNA chip, are for observing the difference of RNA expression between normal cell and abnormal cell. For example, researches for analyzing the difference of RNA expression between human normal cell and cancer cell are performed. Various studies employing high-density oligonucleotide chips, have been performed to analyze the type and system of gene expression of Saccharomyces cerevisea of which total six (6) thousand genes had been fully discovered.
Meanwhile, probes microarrayed on the biochip, are plasmid DNA comprising cDNA, products of polymerase chain reaction or synthetic oligonucleotide.
Probes can be microarrayed on the substrate through photolithography (Affymetrix Inc.), non-contact printing method using Ink-Jet injection process (Piezoelectric printing; Packard Instrument Inc., Syringe-solenoid printing; Cartesian Techonologies), contact printing method (Quill and Splite Pin; Telechem International Inc., Pin and Ring; Genetic Microsystem, Capillary pin; Bioneer Corp.) and so on.
However, whatever microarraying method is used, the amount of probes arrayed in each spot or in each biochip cannot be uniform and also the feature of spot microarrays can not be regular due to the effects caused from types of slide glass, electric noise generated in measuring wavelength and evaporation of spot.
More specifically, the direct synthesis of oligo-type probes on the substrate through photolithography process has critical problem that oligomers thus prepared on the substrate directly are not homogeneous even in same spot due to the failure of synthetic reaction.
Further, the fluctuation of the amount and feature of microarrayed probes may cause critical error especially in interpreting results of observing the difference of RNA expression pattern. The uniformity and regularity of microarrayed probes also should be secured in case of a biochip used for detecting DNA mutation accurately.
Furthermore, the control of the above uniformity and regularity are very important for the research on that DNA mutation or RNA expression should be observed continuously through same probes by using several biochips.
Therefore, the step for controlling the uniformity and regularity of the amount of probes should be necessarily added to a process for manufacturing biochip. In the addition, it is needed to provide the correction factor that indicate such differences between microarrayed for the bio-chip users in order to secure high fidelity of a bio-chip test result through the correction of the hybridization results.
Therefore, the primary object of the present invention is to provide a process of quality examining for microarray of biological material, which comprises a step for mixing probe and a compound which emits light or heat and does not react with said probe, a step for microarraying the mixture obtained in step 1) on a substrate, and a step for measuring light or heat emitted by the scanning of each spots of the microarray mixture.
The other object of the present invention is to provide a microarray of biological material wherein a mixture of probes and a compound which emits light or heat and does not react with said probe, is micrroarryed on substrate.
DISCLOSURE OF THE INVENTION
The object of the present invention is achieved by providing a process of quality examining for microarray of biological material, which comprises:
1) a step for mixing probe and a compound which emits light or heat and does not react with said probe;
2) a step for microarraying the mixture obtained in step 1) on a substrate; and
3) a step for measuring light or heat emitted by the scanning of each spots of the microarray mixture.
In addition, the above method of the present invention may further comprise a step 4) for calibrating the intensity of light or heat emitted from the hybridized microarray of biological material by using the intensity of light or heat measured in said step 3).
Bio-chip users of the present invention can further perform a step for measuring the intensity of light or heat emitted from hybridization of microarrayed probe and target and a step for calculating a correction factor by using the above-measured intensity of light or heat to exclude error caused by discrepancy between size of each spots, in order to get more accurate experimental results.
The other object of the present invention is to provide a microarray of biological material wherein a mixture of probes and a compound which emits light or heat and does not react with said probe is micrroarryed on substrate.
Probes of the present invention are selected from the group consisting of biological materials formed by linear or circular combinations of peptides including amino acid, nucleic acid, polysaccharide and phospholipid, respectively.
In addition, A compound of the present invention which emits light or heat and does not react with the probe is preferably selected from the group consisting of fluorescent material, chemi-luminescent material, bio-luminescent material, calorimetric material and light-scattering material, and is preferably selected from the group consisting of rhodamine derivatives and fluoresceine derivatives including Fluorescein, Coumarin, 4′,5′-Dichloro-2′,7′-dimethoxyfluorescein, Tetramethylrhodamine, X-rhodamine, Eosin, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red and Rodamine and so on, especially in the case that the biological material is nucleic acid.
Two types of rhodamine (6-carboxyrhodamine 6G; 6-CR6G, 5-carboxyrhodamine 6G; 5-CR6G) of the present invention having following structures is activated in 520 nm and emit fluorescence of which wave length is 545 nm.
In addition, the rhodamines do not react with DNA, RNA and protein, and may be washed easily by water or other aqueous solution after measuring the uniformity of microarray of biological material. Therefore, the rhodamines have the advantages that general chip scanner may be used without any extra equipment and subsequent processs including hybridization is not affected thereby.
<Structural Formulas>




FIG. 1 shows a process of quality examining for microarray of biological material by using rhodamine 6G.
More particularly, the predetermined amount of rhodamine is mixed with the predetermined amount of probe, and the mixture is microarrayed. The microarray of the mixture is examined the uniformity of each spot through measuring the intensity of fluorescence, and the result of examination is expressed numerically.
Finally, bio-chip users are provided the numerical data along with a biological chip in order to interpret the result more accurately, in DNA chip research.



BRIEF DESCRIPTION OF THE DRAWINGS
The above objects and other advantages of the present invention will become more apparent by describing in detail a preferred embodiment thereof with reference to the attached drawings, in which:
FIG. 1. shows a process which the results of hybridization are normalized in order to secure high quality of a biological chip.
FIG. 2. shows microarrays wherein oligonucleotides are mixed with rhodamine diluted in the predetermined ratio.
FIG. 3. shows microarrays wherein thirty oligonucleotides (30 mer) are mixed with the same amount of rhodamine.
FIG. 4. shows what effect a process for checking the regularity of the amount of probes has on the results of hybridization.
FIG. 5. shows microarrays which are made after mixtures of oligonucleotide and rhodamine are diluted twice by two fold.
FIG. 6. shows a regressive equation which indicates relationship between fluorescence of microarrays after microarraying and fluorescence of microarrays after hybridization.



BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given for illustration of the invention and not intended to be limiting the present invention.
EXAMPLE 1
Determining the Proper Concentration of Rhodamine 6G for the Purpose of Quality Examining for Microarray of Biological Material
A rhodamine used for obtaining the data to exclude error caused by discrepancy between size of each spot, should meet the following conditions.
First, the concentration of rhodamine should be in the range which can be detected by scanner. That is, the concentration of rhodamine should not too low to be detected and too high not to be saturated.
Second, the rhodamine of the present invention should have the concentration in which rhodamine can be washed easily, with meeting the above condition.
Third, the rhodamine of the present invention should have the concentration in which subsequent process after washing including hybridazation is not affected.
Therefore, the following embodiment was performed in order to determine the most appropriate concentration of rhodamine 6G to meet the above three (3) conditions.
A. Preparation of Rhodamine 6G
10 mM rhodamine 6G was dissolved in methylalchol, and 10 μl of the solution was diluted in 990 μl Sodium Borate buffer (pH9.0; SB buffer) so that the concentration could be 100 pmol/μl.
100 pmol/μl solution was diluted in SB buffer sequentially, and solutions having eleven (11) kinds of concentration are prepared (see FIG. 2).
B. Microarray of the Mixture of Rhodamine and Oligonucleotide
Each diluted solution was mixed with 10 pmol/μl oligonucleotide. Each mixture was microarrayed on a acrylamide gel pad by using HT-ArrayerTM (Bioneer Corp, Korea) by fourteen times (14), respectively. The gel pad was made through smearing 30 μl of the solution composed of 0.1% glycidyl metacrylate, 8% acrylamide, 1/20 ammonium persulfate and 1/100 N,N,N′,N′-tetramethylethylenediamine(TEMED) on a washed slide glass.
C. Scanning and Washing
Rhodamine contained in microarrayed spots was activated in 532nm by using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and was scanned in PMT 600(Photomultipler tubes 600) in order to obtain the fluorescence data.
After that, the chip was washed by water in room temperature for five (5) minutes, and re-scanned in same PMT 600 in same wavelength. The results of scanning were illustrated in FIG. 2.
As illustrated in FIG. 2, 10 pmol/μl oligonuceotide was mixed with rhodamine 6G which was diluted by the predetermined ratio of dilution, and the mixture was microarrayed.
FIG. 2(a) was the result of scanning rhodamine 6G activated in 532 nm, and FIG. 2(b) was the result of scanning in different conditions, for example the condition that a microarray was washed by water in room temperature for five (5) minutes, when increasing the amount of rhodamine which was mixed with oligonucleotide composed thirty (30) monomers.
Rhodamine of which concentration was 1 fmol/μl in lane 1, 5 fmol/μl in lane 2, 10 fmol/μl in lane 3, 50 fmol/μl in lane 4, 100 fmol/μl in lane 5, 500 fmol/μl in lane 6, 1 pmol/μl in lane 7, 5 pmol/μl in lane 8, 10 pmol/μl in lane 9, 50 pmol/μl in lane 10 and 100 pmol/μl in lane 11, was mixed with 10 pmol/μl of oligonucleotide composed of thirty (30) monomers respectively, and each mixture was microarrayed repeatedly fourteen (14) times. The mean diameter of a spot was 180 μm and the mean distance of center to center was 150 μm.
As described in FIG. 2, the intensity of fluorescence was saturated in the case that the concentration of rhodamine was not less than 500 fmol/μl. In addition, the intensity of fluorescence was still detected after washing in the case that the concentration of rhodamine was not less than 1 pmol/μl, because rhodamine was not eliminated through simple washing.
Based on the above results, 100 fmol/μl was selected as the appropriate concentration that the intensity of fluorescence was not saturated and rhodamine was sufficiently eliminated through simple washing for five (5) minutes.
D. Selection of the Washing Condition
It was examined whether 100 fmol/μl rhodamine selected above was eliminated easily by blocking solution (10% ethanolamine) applied generally to a gel pad chip or not.
As illustrated in FIG. 3, 100 fmol/μl rhodamine was mixed with 10 pmol/μl oligonucleotide composed of thirty (30) monomers, and the mixture was microarrayed.
FIG. 3(a) was the results of scanning spots in 532 nm without any treatment after microarraying. FIG. 3(b) was the results of scanning spots in PMT 600 (photomultiplier tubes 600) and FIG. 3(c) was the results of scanning spots in PMT 900 (photomultiplier tubes 900), after washing a microarray by 10% ethanolamine for five (5) minutes.
As results, 100 fmol/μl rhodamine was completely eliminated when washing the microarry by 10% ethanolamine in room temperature for five (5) minutes. That is, no intensity of fluorescence was detected in PMT 600 and in PMT 900. The above results show the advantages of the present invention that an experiment takes less time because a microarry can be washed easily without the addition of extra washing process.
According to the above results, 100 fmol/μl was selected as the most appropriate concentration and washing by water or 10% ethanolamine in room temperature for five (5) minutes was selected as the washing condition.
EXAMPLE 2
The effect of Rhodamine 6G on the Step of Hybridization
Hybridization was performed in order to confirm whether the most appropriate concentration and the washing condition selected in Example 1 were selected properly.
A. Preparation of the Mixture of Rodamine and Oligonucleotide
10 pmol/μl of two types of oligonucleotide was mixed with 100 fmol/μl rhodamine respectively, and the two types of the mixture was mixed with each other sequentially in the ratio from 1:0 to 1.5:8.5 (FIG. 4).
B. Microarray of the Mixture of Rhodamine and Oligonucleotide
Each Mixture was microarrayed on a acrylamide gel pad by using HT-ArrayerTM (Bioneer Corp, Korea) by nineteen (19) times. The gel pad was made through smearing 30 μl of the solution composed of 0.1% glycidyl metacrylate, 8% acrylamide, 1/20 ammonium persulfate and 1/100 N,N,N′,N′-tetramethylethylenediamine(TEMED) on a slide glass.
C. Scanning and Washing
Rhodamine contained in microarrayed spots was activated in 532nm by using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and was scanned in PMT 600(Photomultipler tubes 600) in order to obtain the fluorescence data. After that, the chip was washed by 10% ethanolamine in room temperature for five (5) minutes.
D. Hybridization
Two types of microarrayed oligonucleotide were hybridized with each target that was completely complementary to each oligonucleotide and of which 5′-terminal was labeled by cy3 or cy5. The target labeled by 1 pmol fluorescent material was mixed with 20 μl hybridizing buffer (1M NaCl, 1 mM EDTA, 1% Tween 20 and 5 mM sodium phosphate), and reacted with each oligonucleotide in 40° C. for 1 hour. Non-reacted targets and non-specific products of hybridization were eliminated through washing by hybridizing buffer diluted by ten (10) fold in 65° C. for 15 minutes.
E. Scanning
The intensity of fluorescence of hybridization-product was measured by using a chip scanner (GenePix4000, Axon Instrument Inc, USA), after Cy3 was activated in 532 nm and cy5 was activated in 650 nm.
10 pmol/μl of two types of oligonucleotide (comp-cy3 and comp-cy5) was mixed with each other, and the mixture was mixed with 100 pmol/μl rhodamine, and the final mixture was microarrayed (FIG. 4a).
In addition, the microarray was activated in 532nm, and was washed by ethanolamine in room temperature for five (5) minutes. After washing, the above two (2) kinds of oligonucleotide were hybridized with each target oligonucleotide that was completely complementary to each probe and was labeled by cy3 or cy5.
FIG. 4(b) was the result of measuring the intensity of fluorescence after cy3 was activated in 532nm and cy5 was activated in 635 nm. Green color indicates fluorescence resulted from cy3, and red color indicates fluorescence resulted from cy5, and yellow color indicates the mixture of the above two fluorescence. 10 pmol/μl comp-cy3 and 10 pmol/μl comp-cy5 was mixed with each other sequentially in the ratio from 1:0 to 1.5:8.5, and the mixture was microarrayed in lane 1 to lane 18 of FIG. 4 after mixed with 10 pmol/μl rhodamine.
As results, the feature of yellow color indicating the mixture of two colors by fluorescence corresponded with the mixing ratio of two probe-oligonucleotides.
This results showed that rhodamine using for quality examining of microarray of biological material had no effect on a step for hybridization, especially on a step for competitive hybridization which two targets labeled with two kinds of fluorescent material respectively were hybridized in one spot.
That is, the above results show that rhodamine 6G can be used as the material for quality examining for the microarray of biological material because rhodamine does not cause any error in a process for analyzing genes.
EXAMPLE 3
Exclusion of Error Caused by Discrepancy Between Size of Each Spots
This embodiment was performed in order to show a simple example of calculating a correction factor which can exclude error caused by discrepancy between size of each spots. However, the method for calculating a correction factor is not limited to this embodiment.
A. Preparation of the Mixture of Rodamine and Oligonucleotide
100 pmol/μl of one (1) kind of oligonucleotide was mixed with the same amount of 100 fmol/μl rhodamine 6G. The mixture was diluted in SB buffer by two-fold and by four-fold, and each diluted solution was microarrayed three times (FIG. 5a).
The non-diluted solution was microarrayed in lane 1, lane 2 and lane 3 of FIG. 5. The two-fold diluted solution was microarrayed in lane 4, lane 5 and lane 6, and the four-fold diluted solution was microarrayed in lane 7, lane 8 and lane 9.
B. Scanning and Washing
Rhodamine contained in microarrayed spots was activated in 532nm by using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and was scanned in PMT 600(Photomultipler tubes 600) in order to obtain the fluorescence data. After that, the chip was washed by 10% ethanolamine in room temperature for five (5) minutes.
C. Hybridization
The microarrayed oligonucleotide was hybridized with the target that was completely complementary to the oligonucleotide and of which 5′-terminal was labeled by cy3. The target labeled by 1 pmol fluorescent material was mixed with 20 μl hybridizing buffer (1M NaCl, 1 mM EDTA, 1% Tween 20 and 5 mM sodium phosphate), and reacted with the oligonucleotide in 40° C. for 1 hour. Non-reacted targets and non-specific products of hybridization were eliminated through washing by hybridizing buffer diluted by ten (10) fold in 65° C. for 15 minutes.
D. Scanning
The intensity of fluorescence of hybridization-product was measured by using a chip scanner (GenePix4000, Axon Instrument Inc, USA), after Cy3 was activated in 532nm (FIG. 5b). Table 1 shows the mean intensity of fluorescence obtained respectively after microarraying and after hybridization. Each spot had the area of 7850 μm2, and the semi-diameter of 50 μm.
The intensity of fluorescence of rhodamine obtained after microarraying was linearly proportioned to the concentration of microarrayed oligonucleotide (y=541,63x−12861, R2=0.9453). In addition, the intensity of fluorescence of rhodamine obtained after microarraying highly correlated with the intensity of fluorescence of cy3 obtained after hybridization (R=0.9096).
That is, this results shows that the discrepancy between the amount of microarrayed probes has direct effect on the final hybridization result. Therefore, the final users of a biological chip should be provided the date which can exclude error caused by the above discrepancy in order to correct the final hybridization result. Table 1 shows the mean intensity of fluorescence respectively obtained after microarraying and after hybridization for spots in FIG. 5.
FIG. 6 shows the results of calibrating the regressive equation with respect to the relationship between the intensity of the fluorescence obtained after microarraying and the intensity of the fluorescence obtained after hybridization, by using the mean intensity of fluorescence emitted in the area of 7850μm2 after the intensity of fluorescence in each spot was obtained in 532nm.
The regressive equation was Y(the intensity of fluorescence obtained after hybridization)=2836.6Ln(X;the intensity of fluorescence obtained after microarraying)−18970(R=0.9646).
The intensity of fluorescence of cy3 was inferred from the intensity of fluorescence of rhodamine 6G of which range was 1,000 to 100,000 by using the above regressive equation. The discrepancy between the intensity of fluorescence of rhodamine 6G means the discrepancy between the amount of microarrayed probes, for example oligonucleotide. The intensity of fluorescence of rhodamine 6G of which range was 1,000 to 100,000 can cover almost possible variation thereof.
The arithmetic mean of the intensity values of fluorescence obtained after microarraying was calculated and was converted to the intensity of fluorescence obtained after hybridization (11749.61).
The intensity values of fluorescence obtained after hybridization was subtracted from the value calculated by conversion (11749.61). The values calculated by subtraction were used as correction factors, and the values calculated by adding the correction factor to the actual intensity of fluorescence are always 11749.61.
More particularly, the variation in the amount of probes among spots caused the variation in the intensity of fluorescence of rhodamine, in the case that probes having the predetermined concentration are mixed with 100 fmol/μl rhodamine and the mixture is microarrayed. Finally, the variation in the intensity of fluorescence of rhodamine has the effect on the intensity of fluorescence obtained after hybridization.
For example, assuming that the intensity of fluorescence emitted in spot A is 1000 after microarraying and 1000 after hybridization and the intensity of fluorescence emitted in spot B is 2000 after microarraying and 1500 after hybridization, it can be misunderstood that the amount of hybridized target in spot B is larger than the amount of hybridized target in spot A when judging only by the actual intensity of fluorescence after hybridization.
However, such discrepancy between the intensity of fluorescence can be corrected by subtracting the discrepancy resulting from the amount of microarrayed probes in order to compare the intensity of fluorescence accurately.
In the case that each appropriate correction factor, 11125.07 and 9158.89 is added to each intensity of fluorescence in spot A and in spot B, the intensity of fluorescence is 12125.07 in spot A and 11158.89 in spot B. Therefore, assuming that the same amount of probes is contained in spot A and in spot B, it could be concluded that the amount of hybridized target in spot A is larger than the amount of hybridized target in spot B.
As explained the above, the correction factor can be necessarily used in order to interpret the final results more accurately. Table 2 shows the correction factors calculated by the regressive equation (Y(the intensity of fluorescence obtained after hybridization)=2836.6Ln(X; the intensity of fluorescence obtained after microarraying)−18970).







TABLE 1







The mean intensity of fluorescence according to each


concentration of oligonucleotide after hybridization











the concentration of
the mean intensity of
the mean intensity



microarrayed
fluorescence obtained
of fluorescence



oligonucleotide
in 532 nm after
obtained in 532 nm


No.
(pmole/ul)
microarraying
after hybridization













1
100
50308
11889


2
100
38592
10630


3
100
38254
10848


4
50
12190
8069


5
50
9338
6322


6
50
11395
7952


7
25
3576
5497


8
25
2534
3090


9
25
2423
2232
















TABLE 2







The correction factors calculated by the regressive


equation (Y = 2836.6 Ln(X) − 18970)










the mean
the mean




intensity of
intensity of

the mean normalized


fluorescence
fluorescence

intensity of


obtained in
obtained in

fluorescence obtained


532 nm after
532 nm after
correction
in 532 nm after


microarraying
hybridization
factor
hybridization













1000
624.5386244
11125.07138
11749.61


2000
2590.719917
9158.890083
11749.61


3000
3740.862242
8008.747758
11749.61


4000
4556.901209
7192.708791
11749.61


5000
5189.870207
6559.798798
11749.61


6000
5707.048535
6042.566465
11749.61


7000
6144.307353
5605.302647
11749.61


8000
6525.082501
5226.527499
11749.61


9000
6857.18586
4892.42414
11749.61


10000
7156.051499
4593.558501
11749.61


11000
7426.408355
4323.201645
11749.61


12000
7673.224827
4076.385173
11749.61


13000
7900.273972
3849.336028
11749.61


14000
8110.488846
3639.121354
11749.61


15000
8306.193825
3443.416175
11749.61


16000
8489.263794
3280.346206
11749.61


17000
8661.231596
3088.378404
11749.61


18000
8823.367153
2926.242847
11749.61


19000
8976.734233
2772.875767
11749.61


20000
9122.232792
2627.377208
11749.61


21000
9260.630971
2488.979029
11749.61


22000
9392.589648
2357.020352
11749.61


23000
9518.681517
2230.928483
11749.61


24000
9639.40612
2110.20388
11749.61


25000
9755.201769
1994.408211
11749.61


26000
9866.455264
1883.154736
11749.61


27000
9973.509478
1776.100522
11749.61


28000
10076.66994
1672.940062
11749.61


29000
10176.20998
1573.400024
11749.61


30000
10272.37512
1477.234883
11749.61


31000
10365.38873
1384.223271
11749.61


32000
10455.44509
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11749.61


33000
10542.73197
1206.878027
11749.61


34000
10627.41289
1122.197112
11749.61


35000
10709.63894
1039.971064
11749.61


36000
10789.54845
960.0615548
11749.61


37000
10867.36838
882.3416246
11749.61


38000
10942.91553
806.694475
11749.61


39000
11016.59759
733.0124102
11749.61


40000
11088.41408
661.1959161
11749.61


41000
11158.45715
591.1528512
11749.61


42000
11226.81226
523.7977364
11749.61


43000
11293.55887
456.0511275
11749.61


44000
11358.77094
360.8390601
11749.61


45000
11422.51744
327.0925572
11749.61


46000
11484.86281
264.7471904
11749.61


47000
11545.86731
203.7426886
11749.61


48000
11605.58741
144.0225881
11749.61


49000
11664.07608
85.53391802
11749.61


50000
11721.38306
28.22691844
11749.61


51000
11777.55521
−37.94521415
11749.61


52000
11832.63656
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11749.61


53000
11886.66867
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11749.61


54000
11939.69077
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11749.61


55000
11991.73994
−242.1299376
11749.61


56000
12042.85123
−293.2412303
11749.61


57000
12093.05785
−343.4478507
11749.61


58000
12142.39127
−392.7812681
11749.61


59000
12190.88134
−441.2713877
11749.61


60000
12238.55641
−488.9464096
11749.61


61000
12285.44343
−535.8334275
11749.61


62000
12331.56802
−581.958021
11749.61


63000
12376.95459
−827.3445892
11749.61


64000
12421.62638
−672.0163786
11749.61


65000
12465.60555
−715.9965541
11749.61


66000
12508.91327
−759.3032856
11749.61


67000
12551.56971
−801.9597085
11749.61


68000
12593.59418
−843.9841809
11749.61


69000
12635.00514
−885.3951353
11749.61


70000
12675.82023
−926.210228
11749.61


71000
12716.05636
−953.4463636
11749.61


72000
12755.72974
−1006.119738
11749.61


73000
12794.85588
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11749.61


74000
12833.44967
−1063.839668
11749.61


75000
12871.52543
−1121.915407
11749.61


76000
12909.09682
−1159.486817
11749.61


77000
12946.17708
−1196.567084
11749.61


78000
12982.77888
−1233.168882
11749.61


79000
13018.9144
−1269.304403
11749.61


80000
13054.59538
−1304.985378
11749.61


81000
13089.8331
−1340.323097
11749.61


82000
13124.63844
−1375.028441
11749.61


83000
13159.02189
−1409.411892
11749.61


84000
13192.99356
−1443.383556
11749.61


85000
13226.56318
−1476.953179
11749.61


86000
13259.74016
−1510.130165
11749.61


87000
13292.53359
−1542.923594
11749.61


88000
13324.95223
−1575.342232
11749.61


89000
13357.00455
−1607.394551
11749.61


90000
13388.69874
−1639.088735
11749.61


91000
13420.0427
−1670.432701
11749.61


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INDUSTRIAL APPLICABILITY
As explained the above, final users can be provided a high-quality biological chip through introducing a process of quality examining for microarray of a biological material of the present invention to mass production process of a biological chip. In addition, bio-chip users of the present invention can further perform a step for measuring the intensity of light or heat emitted from hybridized microarray of biological material and a step for calculating a correction factor by using the above-measured intensity of light or heat to exclude error caused by discrepancy between size of each spots, in order to get more accurate experimental results.

## Claims

1. A process for examining the quality of a microarray comprising a biological material, comprising:
providing a substrate;
providing a mixture of a probe and a light-emitting or heat-emitting compound that does not hybridize with the probe and can be removed by washing;
spotting a predetermined amount of the mixture onto each of a plurality of distinct spots on the substrate to produce the microarray;
measuring the amount of light or heat emitted from each spot of the plurality of distinct spots to generate numerical data for each spot;
washing the microarray to remove the light-emitting or heat-emitting compound from each of the plurality of distinct spots; and
for each spot of the plurality of distinct spots, calculating an arithmetic spotting mean of the amount of light or heat emitted from a spot after spotting based on the numerical data.

2. The process of claim 1, further comprising
after removing, contacting the probe contained in each of the plurality of distinct spots with a target molecule comprising a sequence that is completely complementary to the sequence of the probe, wherein the target molecule is labeled with a heat-emitting or light-emitting compound such that upon hybridization of the target molecule with the probe, heat or light is emitted; and
measuring the amount of heat or light emitted from each spot of the plurality of distinct spots to generate numerical hybridization data for each spot.

3. The process of claim 2, further comprising
for each spot of the plurality of distinct spots, calculating an arithmetic hybridization mean of the amount of light or heat emitted from the spot after hybridization based on the numerical hybridization data.

4. The process of claim 3, further comprising
calculating a regression equation from the arithmetic spotting mean for each spot and the arithmetic hybridization mean for each spot; and
calculating a normalized arithmetic hybridization mean of heat or light emitted from each of the plurality of distinct spots from on the regression equation.

5. The process of claim 3, further comprising
calculating a correction factor for each spot of the plurality of distinct spots, each spot having an arithmetic spotted mean corresponding to an amount of probe spotted, the correction factor comprising the difference between the calculated normalized mean and the arithmetic hybridized mean for an arithmetic spotted mean.

6. The process of claim 1, wherein the probe comprises one or more biological materials selected from the group consisting of a nucleic acid, a protein, a polysaccharide, and a phospholipid.

7. The process of claim 6, wherein the nucleic acid comprises a circular or a linear nucleic acid.

8. The process of claim 6, wherein the nucleic acid comprises DNA, cDNA, plasmid DNA, or RNA.

9. The process of claim 1, wherein the light-emitting or heat-emitting compound that does not hybridize with the probe is selected from the group consisting of a fluorescent material, a chemi-luminescent material, a bio-luminescent material, a calorimetric material, and a light-scattering material.

10. The process of claim 9, wherein the biological material is a nucleic acid and the light-emitting or heat-emitting compound is selected from the group consisting of fluorescein; coumarin; 4′,5′-Dichloro-2′,7′-dimethoxyfluorescein;
tetramethylrhodamine; X-rhodamine; eosin; Oregon green; rhodamine green; rhodamine red; Texas red; and Rodamine.

11. The process of claim 9, wherein the biological material is a nucleic acid and the light-emitting or heat-emitting compound is 6-carboxyrhodamine 6G or 5-carboxyrhodamine 6G.