經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（j ) 關連申請案之揭示 臨時申請案序號60/142708，申請日1999年7月7日。 發明背景 1. 發明技術之領域 本發明是有關用於製造DNA晶片而使核酸固定之基 材。此外，本發明也有關於將核酸固定於這樣的基材上之 方法，以及在此基材上偵測標的核酸的方法。 2. 相關技術 由於基因組（genome)分析技術，數種生物體的基因 組結構因而被揭露。與其相比較，基因組功能的分析技術 ，則正在開發中。在這樣的情況下，DNA晶片（DNA微 陣列）是一種引人注目的技術。DNA晶片是一種具有許多 不同基因或片段（DNA片段），固定於一固體基材（例如 ，玻璃載片）表面上之微陣列（microarray)。DNA晶片 (DNA微陣列）可有效於基因表現、突變及多型性（ polymorphism)的分析。 將標的核酸固定於基材上的方法是製造DNA晶片所 需之核心技術。有兩種主要的方法用於製造DNA晶片。一 種方法涉及到核酸在頂端的化學合成，一連接子（linker) 共價鍵結至基材，例如，說明於Science，251卷，767-773 (1991 )以及 Nucleic Acid Research，20 卷，1679-1684 ( I"2)。在這個方法中，DNA是受限於寡核苷酸，並且需 3 本^張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) ' -----------—,—訂---------^9— (請先閱讀背面之注意事項再填寫本頁) 1224619 A7 _ B7 五、發明說明（>〇 要一特別的裝置以控制反應。因此，這不是普遍的方法。 (請先閱讀背面之注意事項再填寫本頁) 另一種方法是將預先合成的DNA或藉由PCR (聚合 酶鏈鎖反應）所製備之DNA(PCR放大的產物），共價地 或非共價地鍵結至基材，例如，說明於Science，270卷， 467-470 ( 1995 )以及 Nucleic Acid Research，22 卷， 5456_5465 ( 1994)。 在非共價的固定方法中，DNA (或RNA)是溶解於鹽 類溶液中，例如標準檸檬酸鹽水（氯化鈉/檸檬酸鈉），同 時伴隨或不具有變性的步驟。將DNA點在玻璃載片上，此 玻璃載片塗覆鹼性的多聚陽離子（例如，聚離胺酸、聚乙 烯亞胺（polyethylenimine))，或以一含有胺基的矽院化 合物（例如，3-胺基丙基三乙氧基砂院）而砍院化，並藉 由紫外光的照射而固定。所有類型的DNA (或RNA)都應 該可藉由這個方法而固定。 經濟部智慧財產局員工消費合作社印製 然而，實務上，寡核苷酸及短的DNA (小於0.3仟鹼 基（kb))，是難以使用這個方法而固定。甚至當長的 DNA (大於0.3仟鹼基）非共價地貼附時，淸洗及雜交（ hybridization)的步驟可將DNA從基材上移除，這可能導 致標的核酸之可偵測度的下降。 非共價固定方法的另一個限制，是增強的背景訊號， 這是由於在基材表面上的殘留鹼性官能基團（陽離子）及 探針核酸（陰離子）之間的非專一性結合所造成的。已知 殘留的胺基基團可藉由乙醯基化（使用酐，例如丁二酸酐 ，而將胺類轉換成羧酸）而被封阻。然而，這個轉換總是 4 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1224619 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（々） 不會完全。 因此，需要發展一種基材，其可應用於標的核酸之超 靈敏偵測，以及適合於許多類型的核酸之固定，包括，短 的及長的核酸、單股核酸、雙股核酸、DNA以及RNA。 發明摘述 本發明的主要目標是：（1)製備使核酸固定之新穎基 材，可以高效率地固定核酸；（2)以核酸固定的基材（也 就是，製備DNA微陣列）；（3)用於固定核酸的製造方 法；以及（4)使用以核酸固定的基材來偵測核酸的方法。 槪要地，本發明主要是有關於使核酸固定之基材，特 別地，是以聚陰離子爲基底的表面，以使核酸貼附。在一 較佳具體實施例中，聚陰離子是聚丙烯酸，然而，任何的 聚陰離子都可以使用。 本發明的另一種形態是涉及到藉由一共價鍵，而將核 酸固定於基材。較佳的方法涉及到將羧酸官能基轉換成活 化的酯類。然後將活化的酯類與胺基修飾的核酸反應。 在第一及第二具體實施例中，基材是一無孔性的表面 。較佳的形式是玻璃。 具體實施例的第三種形態，是有關於以核酸固定的基 材；也就是，以核酸固定的基材，其中，核酸經由聚陰離 子之活化的酯類形式，而共價地鍵結至基材。在第三具體 實施例中，聚陰離子是聚丙烯酸，作爲較佳的形式。較佳 活化的酯類是由聚陰離子及五氟酚（Pentafluorophenol)， 5 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I ϋ 1 ϋ I —m ϋ ϋ ϋ mmmmm I ϋ I ϋ 一SJ I n ϋ I (請先閱讀背面之注意事項再填寫本頁) 1224619 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（今） 藉由碳化二醯亞胺（carbodiimide)偶合反應而製備的五氟 酚酯。 第四具體實施例是有關於將核酸固定至基材的方法； 也就是’使核酸固定至基材的方法，其中，以一聚陰離子 的活化酯類衍生物塗覆之基材，與胺基修飾的核酸接觸。 在第四具體實施例中，聚陰離子是聚丙烯酸，以及基材是 無孔性的，作爲較佳的形式；玻璃是最佳的無孔性基材。 在第四具體實施例中，核酸是以對活化的酯類衍生物 具有反應性的官能基修飾，在較佳的形式中，核酸是以胺 基基團修飾。 第五具體實施例是有關於在試樣（或樣品）中，標的 核酸的偵測方法，包括但並不限於： (a) —種方法，其中用於核酸固定的基材是聚陰離子 之活化的酯類衍生物。聚陰離子先前已共價地貼附到玻璃 載片的表面。 (b) —種在上述活化的酯類衍生物以及核酸之間的反 應’其中核酸是以對於酯類衍生物具有反應性的官能基而 修飾。 (〇 —種在固定於基材上的核酸（標的核酸）以及互 補核酸（探針核酸）之間的雜交方法。 (d) —種用於雜交的核酸之偵測方法。 第六具體實施例是有關於在試樣中之核酸的偵測方法 ，包括但並不限於： (a) —種在基材上之活化的酯類衍生物以及核酸之間 6 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----— — — — — — — — — — — II ^ 1111111 (請先閱讀背面之注意事項再填寫本頁) 1224619 A7 B7Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 B7 V. Description of Invention (j) Disclosure of related applications The provisional application number 60/142708, the application date is July 7, 1999. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a substrate for fixing a nucleic acid for manufacturing a DNA wafer. In addition, the present invention also relates to a method for immobilizing a nucleic acid on such a substrate, and a method for detecting a target nucleic acid on the substrate. 2. Related technologies The genome structure of several organisms has been revealed due to genome analysis techniques. In contrast, analytical techniques for genomic functions are under development. In such cases, DNA chips (DNA microarrays) are a compelling technology. A DNA chip is a microarray with many different genes or fragments (DNA fragments) fixed on the surface of a solid substrate (eg, a glass slide). DNA microarrays (DNA microarrays) are useful for analysis of gene expression, mutations and polymorphism. The method of immobilizing a target nucleic acid on a substrate is a core technology required for manufacturing a DNA wafer. There are two main methods used to make DNA wafers. One method involves the chemical synthesis of a nucleic acid at the top, a linker covalently bonded to the substrate, for example, as described in Science, Vol. 251, 767-773 (1991) and Nucleic Acid Research, Vol. 20, 1679- 1684 (I " 2). In this method, DNA is limited by oligonucleotides and requires 3 copies of the standard to apply Chinese National Standard (CNS) A4 (210 X 297 mm) '----------- —, — Order --------- ^ 9— (Please read the notes on the back before filling this page) 1224619 A7 _ B7 V. Description of the invention (> 〇 A special device is needed to control the reaction. Therefore, this is not a universal method. (Please read the notes on the back before filling this page.) Another method is to use pre-synthesized DNA or DNA prepared by PCR (polymerase chain reaction). ), Covalently or non-covalently bonded to the substrate, for example, as described in Science, Volume 270, 467-470 (1995) and Nucleic Acid Research, Volume 22, 5456_5465 (1994). In the method, DNA (or RNA) is dissolved in a salt solution, such as standard citrate water (sodium chloride / sodium citrate), with or without the step of denaturation. The DNA is spotted on a glass slide. Glass slides are coated with basic polycations (for example, polyionic acid, polyethylenimine), or Silicon compounds containing amine groups (for example, 3-aminopropyltriethoxy sand compound) are chemically modified and fixed by ultraviolet light irradiation. All types of DNA (or RNA) should be available for borrowing. It is fixed by this method. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. However, in practice, oligonucleotides and short DNA (less than 0.3 仟 bases (kb)) are difficult to use this method for fixing. Even when When long DNA (greater than 0.3 bases) is non-covalently attached, the washing and hybridization steps can remove the DNA from the substrate, which may result in a decrease in the detectability of the target nucleic acid. Another limitation of the non-covalent immobilization method is the enhanced background signal, which is caused by the non-specific binding between the residual basic functional group (cation) and the probe nucleic acid (anion) on the substrate surface It is known that residual amine groups can be blocked by acetylation (using anhydrides, such as succinic anhydride to convert amines to carboxylic acids). However, this conversion is always 4 paper sizes Applicable to China National Standard (CNS) A4 specifications ( 210 X 297 mm) 1224619 A7 B7 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. The invention description (发明) will not be complete. Therefore, it is necessary to develop a substrate that can be used for ultra-sensitive detection of target nucleic acids. And suitable for the fixation of many types of nucleic acids, including short and long nucleic acids, single-stranded nucleic acids, double-stranded nucleic acids, DNA and RNA. SUMMARY OF THE INVENTION The main objectives of the present invention are: (1) to prepare novel novel methods for fixing nucleic acids. Substrates that can efficiently immobilize nucleic acids; (2) substrates immobilized with nucleic acids (that is, preparing DNA microarrays); (3) manufacturing methods for immobilizing nucleic acids; and (4) substrates immobilized with nucleic acids Materials to detect nucleic acids. Essentially, the present invention mainly relates to a substrate for immobilizing a nucleic acid, and in particular, a surface based on a polyanion for attaching a nucleic acid. In a preferred embodiment, the polyanion is polyacrylic acid, however, any polyanion can be used. Another aspect of the present invention involves fixing a nucleic acid to a substrate by a covalent bond. The preferred method involves the conversion of carboxylic acid functional groups into activated esters. The activated esters are then reacted with an amino-modified nucleic acid. In the first and second embodiments, the substrate is a non-porous surface. The preferred form is glass. The third aspect of the embodiment relates to a substrate fixed with nucleic acid; that is, a substrate fixed with nucleic acid, in which the nucleic acid is covalently bonded to the base via an activated ester form of a polyanion. material. In a third embodiment, the polyanion is polyacrylic acid as a preferred form. The better activated esters are made of polyanion and pentafluorophenol. 5 paper sizes are applicable to China National Standard (CNS) A4 (210 X 297 mm) I ϋ 1 ϋ I —m ϋ ϋ ϋ mmmmm I ϋ I ϋ I SJ I n ϋ I (Please read the notes on the back before filling in this page) 1224619 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (today) With carbodiimide ( carbodiimide) pentafluorophenol ester. The fourth embodiment relates to a method for fixing a nucleic acid to a substrate; that is, a method for fixing a nucleic acid to a substrate, in which a substrate coated with a polyanionic activated ester derivative and an amine group are used. Modified nucleic acid contact. In a fourth embodiment, the polyanion is polyacrylic acid, and the substrate is non-porous, as a preferred form; glass is the best non-porous substrate. In a fourth embodiment, the nucleic acid is modified with a functional group having reactivity to an activated ester derivative. In a preferred form, the nucleic acid is modified with an amine group. The fifth specific embodiment relates to a method for detecting a target nucleic acid in a sample (or sample), including but not limited to: (a) a method in which a substrate used for nucleic acid immobilization is polyanion activation Ester derivatives. The polyanion has previously been covalently attached to the surface of a glass slide. (b) A reaction between the above-mentioned activated ester derivative and a nucleic acid 'wherein the nucleic acid is modified with a functional group reactive with the ester derivative. (0—A hybridization method between a nucleic acid (a target nucleic acid) and a complementary nucleic acid (a probe nucleic acid) immobilized on a substrate. (D) —A method for detecting a nucleic acid for hybridization. A sixth specific embodiment There are methods for the detection of nucleic acids in samples, including but not limited to: (a) —an activated ester derivative on a substrate and between nucleic acids 6 The paper size applies to Chinese national standards (CNS ) A4 size (210 X 297 mm) -----— — — — — — — — — — — II ^ 1111111 (Please read the precautions on the back before filling this page) 1224619 A7 B7
五、發明說明（< ) 的反應，其中核酸是以對於活化的酯類衍生物具有反應性 的官能基而修飾。 ------------AW (請先閱讀背面之注意事項再填寫本頁) (b) —種在固定於基材上的核酸以及互補核酸之間的 雜交方法。 (C) 一種用於雜交的核酸之偵測方法。 發明詳述 聚陰離子是一含有兩個或多個負電荷的聚合物。 名詞“核酸”包括去氧核糖核酸（DNA)及核糖核酸 (RNA)，其以寡核苷酸信使RNA (mRNA)、反義、質 體DNA、質體DNA的部份或衍生自病毒（病毒DNA)線 形DNA的遺傳物質、或染色體DNA的形式存在。 淫度恆定裝置是指任何可維持一固定溼度範圍的腔室 〇 在本發明中，用於使核酸固定之基材並無限制，只要 可應用於DNA晶片或生物感測器的基材都可以使用。基材 包含一具有無孔性及平滑表面的固體支持物，例如，以較 佳形式的玻璃載片或二氧化矽珠是極佳可使用的材料。 經濟部智慧財產局員工消費合作社印製 在一具體實施例中，基材是以3-胺基丙基三乙氧基矽 烷處理，以提供具有可與聚陰離子反應的官能基之基材表 面（在這個實例中是胺基）。任何可將官能基導入至玻璃 基材表面上的矽烷，都可使用於本發明中。然後聚陰離子 可藉由碳化二醯亞胺的偶合反應，而共價地貼附至基材之 矽烷化的表面。 7 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1224619 經濟部智慧財產局員工消費合作社印製 A7 ___B7____ 五、發明說明（b ) 聚陰離子包含酸性的官能基團’例如，羧酸根、磷酸 根、硫酸根等等，其具有避免與探針核酸非專一性鍵結（ 靜電）的效果。聚陰離子共價地貼附至基材的表面；聚丙 烯酸、聚麩胺酸、聚天門冬胺酸以及聚磷酸根是較佳的形 .式。這些聚陰離子經由碳化二醯亞胺（或其他活化的酯類 )化學反應，而貼附至基材之矽烷化的表面。 聚陰離子之酸性的官能基團必須活化（形成一活化的 酯類），以將標的核酸固定在聚陰離子塗覆之基材上。例 如，五氟酚及咪唑酯類分別是羧酸根及磷酸根可用之活化 的酯類衍生物。 對於將核酸固定在基材上並無特定的限制。所有的寡 核苷酸、聚核苷酸及其衍生物都可以是有用的。單股及雙 股形式的核酸（DNA或RNA)也是有效的。衍生物並沒有 任何限制，只要它們具有可固定至基材表面的修飾基即可 。在DNA的5’端以胺基、硫醇基、磷酸根及醛基修飾的 衍生物可作爲實例。此外，在5’端以一交聯劑（cross linker)或連接子（linker)作爲間隔（例如，烷基胺）而 修飾的衍生物，也是有效的。在本發明中，在5’端的修飾 是特別有效於短的核酸（例如，寡核苷酸）之固定。也可 使用任何共價修飾以導入各種官能基或訊號基團的核酸， 可使用Mirus’ Label IT試劑以修飾核酸。 上述修飾的核酸是溶解在適當的固定化溶液中’例如 ，2〇 mM (毫莫耳濃度）的MOPS ( 3-嗎啉丙烷硫酸鹽）緩 衝液或50 mM碳酸鹽緩衝液（pH 9_5)，濃度在0·〇1-2·0 8 本張尺度適用中關家鮮（CNS)A4規格（210 X 297公釐) """" 一 丨丨丨丨丨i — ！ I -訂·-11 — -1 (請先閱讀背面之注意事項再填寫本頁) 1224619 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明說明（”） 毫克/毫升，較佳地在0.1-1.0毫克/毫升。同時伴隨或不具 有變性的步驟，所需要的核酸可藉由接觸，而固定至具 活化的酯類官能基表面處理的基材上。也就是，使用微定 量吸管或DNA晶片製備裝置（DNA陣列儀），上述之固 定量的DNA溶液，可點在上述具有活化的酯類官能基表面 處理的基材上。將其保持在溼度恆定裝置中30-60分鐘， 並分別以2%SDS (硫酸十二酯鈉）及蒸餾水淸洗。此外， 如果需要的話，可將固定有核酸的基材在室溫下浸泡在0.3 Μ氫氧化鈉中5分鐘，或浸泡在沸水中2分鐘，以使核酸 變性，然後以蒸餾水及無水酒精淸洗並乾燥。藉由這個處 理，未與核酸反應之活化的酯類基團，被水解回復到竣酸 的分子部份（也就是，負電荷）。酸性基團可避免在探針 核酸及基材之間的非專一性靜電鍵結。一般而言，可省略 對聚離胺酸載片是必要的丁二酸酐封阻步驟。這些酸性基 團也可降低背景訊號。 固定在基材上的核酸之品質，可藉由使用螢光標示的 核酸而測定。標示的標的核酸可被共價地固定，並且殘留 的螢光訊號可被偵測。本發明的固定方法，可在基因中超 靈敏地偵測表現的程度、突變、多型性等，因爲這個方法 提供高效率的固定化作用。此外，已固定的核酸，在雜交 的條件下將不會脫離（因爲核酸被共價地貼附至基材）。 使用一般的雜交技術，以偵測共價地貼附至基材（例 如，DNA晶片）之標的核酸。探針核酸是以一螢光原（或 其他的訊號基團）標示，並且藉由鹼或熱而變性。將標示 9 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） ------------------- 訂---------· (請先閱讀背面之注意事項再填寫本頁) 1224619 A7 B7 五、發明說明（2 ) 的探針核酸加到雜交溶液中。將雜交溶液中的5微升到2〇 微升部份滴在陣列的表面上，以一蓋玻片小心覆蓋，以避 免空氣氣泡在蓋玻片底下形成。將其以適當的溫度及時間 ，保持在溼度恆定裝置中。移除蓋玻片，將載片徹底淸洗 ，移除溼氣，最後並藉由螢光掃描器（螢光判讚器）偵測 螢光訊號。 在本發明中所特定的聚陰離子塗覆之玻璃載片’是可 以有效地固定核酸。藉由共價貼附的聚陰離子’在基材表 面上所形成的立體基質，可提供增加數量的核酸之固定。 在本發明中之基材以及固定的核酸，提供超靈敏的核 酸偵測訊號以及低的背景訊號偵測。因爲當活化的酯類被 水解時，未與標的核酸反應之聚陰離子的活化酯類衍生物 可回復成聚陰離子，因此可抑制負電荷的探針核酸’非專 一性的鍵結至基材。 使用本發明之方法可達到標的核酸偵測之增進的靈敏 度，結果··（1)標的核酸（包括寡核苷酸）高效率地貼附 至基材；（2)在固定之後，可省略封阻的步驟；（3)在 標的核酸及基材之間的非專一性鍵結，可保持在最小量； 以及（4)聚陰離子處理的立體效果可提供高的雜交效率。 實施例 我們藉由以下的實施例而淸楚地解釋本發明。然而， 本發明並不限制於這些明確的實施例。 10 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------ΑΨ (請先閱讀背面之注意事項再填寫本頁) tr---------ΜΨ. 經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（f ) 實施例（說明）1 (1)在序列表中分別顯示爲序列編號：1及序列編號 :2的兩種噬菌體DNA片段（分別是ι·〇仟鹼基及0.3仟 鹼基，後者是前者的一部份），是藉由聚合酶鏈鎖反應（ PCR)而製備，以作爲一標準的DNA。 PCR是使用噬菌體DNA作爲模板，以及以下的兩個 引子組合而進行；在序列表中，分別顯示爲序列編號：3 和序列編號：4的引子S和引子Α1000，以及引子S和序 列編號：5的引子Α300。之後，使用QiaqUick PCR純化套 組試劑96(Qiager〇，根據使用手冊而純化每個放大的片 段。使用蒸餾水以將DNA從管柱中沖提出來。DNA藉由 冷凍乾燥而濃縮。 將每個DNA片段溶解在20 mM MOPS緩衝液（pH 7.5 )中，陣歹丨J是藉由GMS 417陣列儀（Genetic Microsystem)，而製作在具有胺基在其表面的玻璃載片上 ，這種玻璃載片是商業上可獲得的。所使用的玻璃載片是 聚-L-離胺酸塗覆的P〇LY_PREP-SLIDES (Sigma;簡寫爲 PLL塗覆的玻璃載片），以及MAS (胺基烷基矽烷家族） 塗覆的載片（Matsunami Glass Ind.;簡寫爲MAS塗覆的玻 璃載片）。這些載片是廣泛地使用於DNA晶片的製備。這 些點陣（spots)是分別以150微米的大小及375微米的間 距而建立。配置相同DNA的5個點陣。 在DNA點上之後，將每個載片保持在控制於37。(：及 90%溼度的培養箱中1小時，然後以60毫焦耳/平方公分 11 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------ (請先閱讀背面之注意事項再填寫本頁) tr--------- 經濟部智慧財產局員工消費合作社印製 1224619 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（、D ) 進行紫外線交聯反應。載片以0.2%硫酸十二酯鈉淸洗一次 ，並以蒸館水淸洗兩次，藉由低速（大約1，〇〇〇轉/分鐘） 離心而移除溼氣，然後風乾。將上述預先處理的載片以蒸 館水徹底淸洗，在水浴中煮沸3分鐘，並暴露至冰冷的乙 醇中以使DNA變性。藉由低速離心而移除乙醇。之後，將 載片風乾並儲存在乾燥箱中。 將一部份載片以無水丁二酸酐處理，藉由丁二醯基化 作用而封阻在基材表面上的游離胺基。封阻溶液是藉由將 溶於89_5笔升N-甲基-2-卩比略院酮的1.5克丁二酸酐，與9 毫升1 Μ的硼酸鹽緩衝液（pH 8.0)混合而製備。將載片 在室溫下，浸泡在封阻溶液中20分鐘。將處理的載片以蒸 餾水徹底淸洗，在水浴中煮沸3分鐘，並暴露至冰冷的乙 醇中以使DNA變性。藉由低速離心而移除乙醇。之後，將 載片風乾並儲存在乾燥箱中。 (2 )將商業上可獲得的胺基院基砂院玻璃載片（原位 PCR玻璃載片，PE Biosystems ;簡寫爲AAS載片玻璃） ，浸泡在2.5%的戊二醛溶液中1小時，以蒸餾水淸洗三次 ，然後風乾。產生一塗覆有醛基基團的載片。 在序列表中顯示爲序列編號：6之24-聚物的寡核苷酸 ，與鏈長6的烷基胺基連接子（PE Biosystems)，在5’端 鍵結（簡寫爲NH2-F Oligo)，其是在DNA合成儀上製備 。接著，將它溶解在20 mM MOPS緩衝液（pH 7.5 )中， 至最終濃度爲10 mM。將這個合成的DNA使用GMS 417 陣列儀，以相同於在實施例說明1- ( 1 )中所述的方式， 12 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -έ— ·ϋ 1« a·— 1· IHI MM H·· MM 麵 _ 言 (請先閱讀背面之注意事項再填寫本頁) 1224619 A7 B7____ 五、發明說明（\\) (請先閱讀背面之注意事項再填寫本頁) 點在戊二醛處理的載片上。其後，將載片玻璃以0.2%的 SDS淸洗2分鐘，並持續地以蒸餾水淸洗。爲了減少席夫 鹼基（Schiff-base)變爲穩定的胺（席夫鹼基是藉由在基 材上的醛基及在DNA上的胺基之間，共價鍵結所形成的） ，因此將載片浸泡在含有磷酸鹽緩衝液（PBS) -100%酒 精（體積比3 : 1)之0.25%氫硼化鈉中5分鐘，然後風乾 〇 (3) 聚陰離子共價貼附至固體支持物： 經濟部智慧財產局員工消費合作社印製 將玻璃顯微鏡載片分別在2 Μ硝酸、2 Μ氫氧化鈉、 純水以及丙酮中，藉由超音波震盪而淸潔（在每個溶液中 進行10分鐘）。將淸潔的載片，以溶解在95%酒精中的4 %之3_胺基丙基三乙氧基矽烷，而胺基矽烷化2分鐘，然 後在l〇〇°C中培養10分鐘，以將其表面胺基丙基矽烷化。 將聚丙烯酸溶解在二甲基甲醯胺中，至最終濃度爲5毫克/ 毫升，此外，加入1-[(3-二甲基胺基）丙基>3-乙基碳化二 醯亞胺氫氯化物（簡寫爲EDC)，至最終濃度爲4.0克 /100毫升。將胺基矽烷化的載片浸泡在這個溶液中隔夜， 以二甲基甲醯胺、甲醇、純水以及丙酮潤洗。將聚丙烯酸 塗覆的載片在真空下乾燥。 (4) 聚陰離子塗覆的固體支持物之活化 將聚丙烯酸塗覆的載片以下列的方式、轉變成活化的 酯類所塗覆的載片。將1.8克五氟酚、2_〇克EDC以及13 毫克二甲基胺基毗啶溶解在50毫升的二甲基甲醯胺中。由 實施例說明^ (3)所製備之聚丙烯酸塗覆的載片’浸泡 13 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 1224619 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（丨/) 在這個溶液中5小時，以二甲基甲醯胺及二氯甲烷潤洗， 在真空下乾燥，最後並儲存在乾燥箱中。 實施例（說明）2 (1) 將兩種大小的λ噬菌體DNA片段（1.0仟鹼基 及0.3仟鹼基），藉由與實施例1-(1)中相同的方法而製 備。在這個合成中，在末端具有胺基的DNA片段，藉由使 用含有引子S、並與鏈長6的烷基胺基連接子（ΡΕ Biosystems)在5’端鍵結的胺基而放大。純化之後，將每 個含有胺基的DNA片段溶解在20 mM MOPS緩衝液（pH 7.5)中，至最終濃度爲0.5毫克/毫升，然後以三重複的方 式，點在由實施例1- (4)之方法所活化之活化的酯類所 塗覆的玻璃載片上。作爲對照組，在PLL塗覆的載片玻璃 上製備相同的陣列。 以DNA點上之活化的酯類所塗覆的玻璃載片，保持 於控制在37°C及90%溼度的培養箱（Tokyo Rikakikai: Eycla型號KC-1000)中30分鐘，接著在室溫下培養在0.2 %SDS中，以洗掉過量的鹽，然後再以蒸餾水淸洗。之後 ，將載片浸泡在0.3 N氫氧化鈉中5分鐘，以使DNA變性 ，再以蒸餾水徹底淸洗，藉由在大約1，〇〇〇轉/分鐘的轉速 離心而乾燥，並儲存在乾燥箱中。 (2) 雜交反應 在每個玻璃載片上製備的核酸陣列’以一標示有螢光 原的核酸探針雜交並觀察之。將每個DNA陣列在含有50 14 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) ------1--1 ---— II--訂- - ----1 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（Y>1 ) %甲醯胺、0.2%SDS、5x Denfardt’溶液、〇·1毫克/毫升鮭 魚精子DNA以及6χ標準檸檬酸鹽（SSC)的預先雜交緩 衝液中，以42°C預先雜交1小時。 將實施例1- ( 1 )中製備的1.0仟鹼基λ噬菌體DNA .片段，藉由使用Label IT® Cy5TM標示套組試劑（Minis)， 根據使用手冊而以Cy5TM標示。將標示的核酸藉由酒精沈 澱而純化。以預先雜交緩衝液稀釋這個Cy5TM標示的核酸 探針，至最終濃度爲20、2及0.2奈克(ng)/毫升，在95°C 加熱2分鐘，並冷卻至室溫。將不溶的物質藉由離心而移 除。將大約5微升的雜交溶液滴在核酸陣列上。小心覆蓋 一層蓋玻片，以避免產生氣泡，並將其在溼度恆定裝置中 ，以42°C培養20小時。將蓋玻片在室溫下，在2x SSC溶 液中移除，以含有2x SSC的0.2%SDS，在65°C淸洗5分 鐘兩次，以及在55°C淸洗30分鐘。然後，將其在室溫下 ，以0.05x SSC淸洗10分鐘，藉由在大約1，000轉/分鐘的 轉速離心而移除溼氣，然後風乾。 雜交之後，核酸陣列藉由使用具有635 nm發散波長 以及660 nm激發波長的GMS 418陣列掃描儀（GMS)而 判讀。偵測的訊號強度藉由影像分析軟體ImaGene ( Biodiscovery)而分析。所得到的結果摘述於表1中。 15 本紙張尺度適用中國國豕標準（CNS)A4規格（21〇 X 297公釐） ϋ ·ϋ I —Bi I n i^i · mmmmm ϋ I. n _ϋ· —Bi ϋ I _ tl (請先閱讀背面之注意事項再填寫本頁) 1224619 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明 表1 探針 固定的DNA片段 用於DNA固定之基材 濃度 大小 活化的酯類塗覆的 PLL塗覆的 (奈克/毫升） (鹼基對） —__片玻璃 載片玻璃 0.2 1000 1484 889 300 988 856 2 1000 6640 2702 300 2635 1077 20 1000 27383 14310 300 7099 3824 如表1所示，在所有的探針濃度中，在活化的酯類塗 覆的玻璃載片上之每個大小的固定化DNA，其訊號均大於 PLL塗覆的玻璃載片上所產生的訊號。也就是，在標的 PCR放大的核酸片段之偵測中，顯示使用由活化的酯類塗 覆的玻璃載片所製備的DNA晶片，可較常用的（PLL)塗 覆的玻璃載片所製備的DNA晶片，提供較高的訊號。 實施例（說明）3 (1 )具有寡核苷酸-DNA陣列的DNA晶片之構築 在DNA合成儀上製備兩條24個核苷酸長度的寡核芽 酸；在序列表中，寡核苷酸顯示爲序列編號：6 ’其中一條 寡核苷酸以鏈長6的烷基胺基連揆子（PE Biosystems) ’ 在5,端修飾（簡寫爲NH2-F 〇lig〇) ’以及另一條寡核芽 酸是在5,端修飾，以包含一羥基棊團（簡寫爲0H_F 01ig〇 16 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I ^1 ^1 1>1 .^1 ^1 ϋ ϋ n ϋ I ϋ ϋ I ϋ I 一ej -ϋ I ϋ ϋ ϋ ϋ ϋ I (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（V< ) )。也合成另兩條24個核苷酸長度的寡核苷酸；其中一條 寡核苦酸具有與顯不爲序列編號：7之第一寡核苦酸互補 的序列，在5’端具有Cy3TM (Cy3—R 01igo);以及另一條 寡核苷酸具有與顯示爲序列編號：14無關之序列，在5’端 具有烷基胺基連接子（NH2-N01igo)。 將 OH-F Oligo、NH2_F Oligo 以及 NH2-N Oligo 溶解在 20 mM MOPS緩衝液（pH 7.5)中，至最終濃度爲5、50 及500//Μ，然後配置在由實施例1- (4)的方法所活化之 活化的酯類塗覆的玻璃載片上。在那時，每個DNA序列配 置7個點。點上去之後，將載片如實施例2中所說明的方 式淸洗並乾燥，最後將其處理成爲寡核苷酸晶片。作爲對 照組，在戊二醛處理的載片上（如實施例1- (2)中所述 )製備相同的寡核苷酸陣列。 (2)寡核苷酸晶片的雜交反應5. The reaction of the invention description (<), wherein the nucleic acid is modified with a functional group having reactivity to an activated ester derivative. ------------ AW (Please read the notes on the back before filling out this page) (b) — A hybridization method between the nucleic acid fixed on the substrate and the complementary nucleic acid. (C) A method for detecting nucleic acids for hybridization. DETAILED DESCRIPTION A polyanion is a polymer containing two or more negative charges. The term "nucleic acid" includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are derived from oligonucleotide messenger RNA (mRNA), antisense, plastid DNA, plastid DNA, or derived from a virus (virus DNA) exists in the form of genetic material of linear DNA or chromosomal DNA. A constant-incidence device refers to any chamber that can maintain a fixed humidity range. In the present invention, there is no restriction on the substrate for fixing nucleic acids, as long as it can be applied to a substrate of a DNA wafer or a biosensor use. The substrate contains a solid support having a non-porous and smooth surface. For example, glass slides or silica beads in a better form are excellent materials that can be used. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economy In this example it is amine). Any silane that can introduce a functional group onto the surface of a glass substrate can be used in the present invention. The polyanion can then be covalently attached to the silanized surface of the substrate by a coupling reaction of carbodiimide. 7 This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1224619 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 ___B7____ V. Description of the invention (b) The polyanion contains acidic functional groups. , Carboxylate, phosphate, sulfate, etc., which has the effect of avoiding non-specific binding (static) with the probe nucleic acid. Polyanions are covalently attached to the surface of the substrate; polyacrylic acid, polyglutamic acid, polyaspartic acid, and polyphosphate are the preferred forms. These polyanions are attached to the silanized surface of the substrate via a chemical reaction of carbodiimide (or other activated esters). The acidic functional group of the polyanion must be activated (forming an activated ester) to immobilize the target nucleic acid on the polyanion-coated substrate. For example, pentafluorophenol and imidazole esters are activated ester derivatives of carboxylate and phosphate, respectively. There are no specific restrictions on the immobilization of nucleic acids on a substrate. All oligonucleotides, polynucleotides and their derivatives can be useful. Nucleic acids (DNA or RNA) in single and double strand form are also effective. The derivatives are not limited as long as they have a modifying group that can be fixed to the surface of the substrate. Derivatives modified with an amine group, a thiol group, a phosphate group, and an aldehyde group at the 5 'end of DNA are examples. In addition, derivatives modified at the 5 'end with a cross linker or linker as a spacer (for example, an alkylamine) are also effective. In the present invention, the modification at the 5 'end is particularly effective for fixing short nucleic acids (e.g., oligonucleotides). Any covalently modified nucleic acid can be used to introduce various functional groups or signal groups, and the Mirus' Label IT reagent can be used to modify the nucleic acid. The above-mentioned modified nucleic acid is dissolved in an appropriate immobilization solution ', for example, a 20 mM (millimolar concentration) MOPS (3-morpholine propane sulfate) buffer or a 50 mM carbonate buffer (pH 9_5), Concentration is 0 · 〇1-2 · 0 8 This scale is applicable to Zhongguan Jiaxian (CNS) A4 specification (210 X 297 mm) " " " " 丨 丨 丨 丨 丨 i —! I -Order · -11 — -1 (Please read the notes on the back before filling this page) 1224619 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Α7 Β7 V. Description of Invention (") mg / ml, preferably in 0.1-1.0 mg / ml. At the same time with or without denaturation step, the required nucleic acid can be fixed to the substrate with activated ester functional surface treatment by contact. That is, using a micro-quantitative pipette or DNA wafer preparation device (DNA array analyzer), the above-mentioned fixed amount of DNA solution can be spotted on the above-mentioned substrate with activated ester functional surface treatment. Keep it in a constant humidity device for 30-60 minutes, and Rinse with 2% SDS (sodium lauryl sulfate) and distilled water. In addition, if necessary, the substrate with the nucleic acid immobilized can be immersed in 0.3 M sodium hydroxide for 5 minutes at room temperature, or immersed in boiling water For 2 minutes to denature the nucleic acid, then rinse with distilled water and absolute alcohol and dry. By this treatment, the activated ester groups that have not reacted with the nucleic acid are hydrolyzed back to the molecular part of the acid (that is, , (Negative charge). The acidic group can avoid non-specific electrostatic bonding between the probe nucleic acid and the substrate. In general, the succinic anhydride blocking steps necessary for polyionine slides can be omitted. These Acidic groups can also reduce background signals. The quality of nucleic acids immobilized on a substrate can be determined by using fluorescently labeled nucleic acids. The labeled target nucleic acids can be covalently immobilized, and residual fluorescent signals can be detected. Detection. The immobilization method of the present invention can super sensitively detect the degree of expression, mutation, polymorphism, etc. in the gene, because this method provides high-efficiency immobilization. In addition, the immobilized nucleic acid can be used under hybridization conditions. Will not detach (because the nucleic acid is covalently attached to the substrate). Use common hybridization techniques to detect the target nucleic acid that is covalently attached to the substrate (for example, a DNA wafer). Probe nucleic acids are Labeled with a fluorescein (or other signal group), and denatured by alkali or heat. 9 paper sizes will be marked. Applicable to China National Standard (CNS) A4 (210 X 297 public love) ---- --------------- order- -------- · (Please read the precautions on the back before filling out this page) 1224619 A7 B7 V. Description of the invention (2) The probe nucleic acid is added to the hybridization solution. 5 microliters of the hybridization solution Drop 20 microliters on the surface of the array and carefully cover it with a coverslip to avoid the formation of air bubbles under the coverslip. Keep it in a constant humidity device at an appropriate temperature and time. Remove the coverslip, rinse the slide thoroughly, remove the moisture, and finally detect the fluorescent signal by a fluorescent scanner (fluorescent judgement). The polyanion coated in the present invention Glass slides can effectively fix nucleic acids. The three-dimensional matrix formed on the surface of the substrate by covalently attached polyanions can provide an increased amount of nucleic acid fixation. The substrate and the fixed nucleic acid in the present invention provide ultra-sensitive nucleic acid detection signals and low background signal detection. Since the activated ester derivative of a polyanion which has not reacted with the target nucleic acid can be restored to a polyanion when the activated ester is hydrolyzed, the non-specific binding of the negatively charged probe nucleic acid to the substrate can be suppressed. The method of the present invention can achieve the enhanced sensitivity of target nucleic acid detection. As a result, (1) the target nucleic acid (including oligonucleotides) is efficiently attached to the substrate; (2) the sealing can be omitted after fixing (3) non-specific bonding between the target nucleic acid and the substrate, which can be kept to a minimum; and (4) the three-dimensional effect of the polyanion treatment can provide high hybridization efficiency. EXAMPLES The present invention is explained in detail by the following examples. However, the present invention is not limited to these specific examples. 10 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ----------- ΑΨ (Please read the precautions on the back before filling this page) tr ---- ----- ΜΨ. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 B7 V. Description of the Invention (f) Example (Explanation) 1 (1) The serial number is displayed in the sequence list: 1 and the serial number : The two phage DNA fragments (2 are ι · 〇 仟 bases and 0.3 仟 bases, the latter are part of the former), were prepared by polymerase chain reaction (PCR) as a standard Of DNA. PCR is performed using phage DNA as a template and the following two primers are combined; in the sequence listing, the sequence number: 3 and sequence number: 4 are the primer S and primer A1000, and the primer S and sequence number: 5 The primer A300. After that, each amplified fragment was purified using QiaqUick PCR purification kit 96 (Qiager0, according to the instruction manual. Distilled water was used to purge the DNA from the column. The DNA was concentrated by freeze drying. Each DNA was concentrated The fragment was dissolved in 20 mM MOPS buffer (pH 7.5). The array was prepared on a glass slide with an amine group on its surface by a GMS 417 array instrument (Genetic Microsystem). This glass slide is Commercially available. The glass slides used are poly-L-lysine-coated POLY_PREP-SLIDES (Sigma; abbreviated as PLL-coated glass slides), and MAS (aminoalkylsilane (Family) coated slides (Matsunami Glass Ind .; abbreviated as MAS-coated glass slides). These slides are widely used in the preparation of DNA wafers. These spots are each 150 microns in size And 375 micron spacing. 5 dots of the same DNA were allocated. After the DNA spots, each slide was kept at 37. (: and 90% humidity incubator for 1 hour, then 60 11 millijoules per square centimeter Home Standard (CNS) A4 Specification (210 X 297 mm) ------------ (Please read the precautions on the back before filling this page) tr --------- Economy Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Education 1224619 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (, D) UV-crosslinking reaction. Rinse twice with steamed water, remove the moisture by centrifugation at a low speed (about 10,000 rpm), and then air-dry. The above-prepared slides are rinsed thoroughly with steamed water. Boil in a water bath for 3 minutes and expose to ice-cold ethanol to denature the DNA. The ethanol was removed by low-speed centrifugation. After that, the slides were air-dried and stored in a dry box. A portion of the slides were dried with anhydrous diethyl ether. Anhydride treatment blocked the free amine groups on the surface of the substrate by succinyl hydration. The blocking solution was made by dissolving 1.5-5 N-methyl-2-pyrrolidone in 89_5 pen liters. G of succinic anhydride was prepared by mixing with 9 ml of 1 M borate buffer (pH 8.0). The slide was immersed in room temperature at The solution was blocked in the solution for 20 minutes. The treated slide was thoroughly rinsed with distilled water, boiled in a water bath for 3 minutes, and exposed to ice-cold ethanol to denature the DNA. The ethanol was removed by low-speed centrifugation. After that, the slide was Air-dried and stored in a dry box. (2) Commercially available amine-based glass sand glass slides (in-situ PCR glass slides, PE Biosystems; abbreviated as AAS slide glass), immersed in 2.5% The glutaraldehyde solution was rinsed three times with distilled water for 1 hour, and then air-dried. A slide coated with aldehyde groups was produced. Shown in the sequence listing as a sequence number: 24-mer oligonucleotide of 6 and 6-chain alkylamine linker (PE Biosystems), bonded at the 5 'end (abbreviated as NH2-F Oligo ), Which was prepared on a DNA synthesizer. Next, it was dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 10 mM. Using this synthetic DNA with a GMS 417 arrayer, in the same manner as described in Example Explanation 1- (1), 12 paper sizes are applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm)- έ— · ϋ 1 «a · — 1 · IHI MM H ·· MM face _ words (please read the precautions on the back before filling this page) 1224619 A7 B7____ 5. Description of the invention (\\) (Please read the back on the first Note again on this page) Click on the glutaraldehyde-treated slide. Thereafter, the slide glass was rinsed with 0.2% SDS for 2 minutes, and continuously rinsed with distilled water. In order to reduce the Schiff-base into a stable amine (Schiff-base is formed by covalent bonding between the aldehyde group on the substrate and the amine group on the DNA), Therefore, the slide was immersed in 0.25% sodium borohydride containing phosphate buffered saline (PBS)-100% alcohol (3: 1 by volume) for 5 minutes, and then air-dried. (3) Polyanion was covalently attached to the solid Supports: Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, glass microscope slides were cleaned in 2 mM nitric acid, 2 mM sodium hydroxide, pure water, and acetone, and cleaned by ultrasonic vibration (in each solution For 10 minutes). Clean the slide to 3% aminopropyltriethoxysilane in 4% dissolved in 95% alcohol, while the aminosilane is alkylated for 2 minutes, and then incubated at 100 ° C for 10 minutes, In order to silamine its surface. Polyacrylic acid was dissolved in dimethylformamide to a final concentration of 5 mg / ml, and 1-[(3-dimethylamino) propyl> 3-ethylcarbodiimide was added Hydrochloride (abbreviated as EDC) to a final concentration of 4.0 g / 100 ml. Soak the aminosilanized slide in this solution overnight and rinse with dimethylformamide, methanol, pure water, and acetone. The polyacrylic-coated slide was dried under vacuum. (4) Activation of polyanion-coated solid support The polyacrylic-coated slide was converted into an activated ester-coated slide in the following manner. 1.8 g of pentafluorophenol, 2.0 g of EDC and 13 mg of dimethylaminopyridine were dissolved in 50 ml of dimethylformamide. Explained by Example ^ (3) Polyacrylic-coated slide prepared 'Immersion 13' This paper size is applicable to China National Standard (CNS) A4 (210 x 297 mm) 1224619 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Preparation of A7 B7 V. Description of the invention (丨 /) In this solution for 5 hours, rinse with dimethylformamide and dichloromethane, dry under vacuum, and finally store in a drying box. Example (Explanation) 2 (1) Two kinds of λ phage DNA fragments (1.0 仟 base and 0.3 仟 base) were prepared by the same method as in Example 1- (1). In this synthesis, a DNA fragment having an amine group at the terminal end was amplified by using an amine group having a primer S and an amine group bonded to the 5 'end of an alkylamine linker (PE Biosystems) having a chain length of 6. After purification, each amine-containing DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 0.5 mg / ml, and then repeated in a triplicate manner, as described in Example 1- (4 ) On glass slides coated with activated esters. As a control group, the same array was prepared on a PLL-coated slide glass. Glass slides coated with DNA-activated esters were kept in an incubator (Tokyo Rikakikai: Eycla Model KC-1000) controlled at 37 ° C and 90% humidity for 30 minutes, then at room temperature Cultivate in 0.2% SDS to wash away excess salt, then rinse with distilled water. Thereafter, the slides were immersed in 0.3 N sodium hydroxide for 5 minutes to denature the DNA, and then rinsed thoroughly with distilled water, dried by centrifugation at a speed of about 1,000 rpm, and stored in a dry state. In the box. (2) Hybridization reaction The nucleic acid array 'prepared on each glass slide was hybridized with a nucleic acid probe labeled with a fluorogen and observed. Each of the DNA arrays contained 50 14 paper standards that were in accordance with Chinese National Standard (CNS) A4 specifications (210 X 297 mm) ------ 1--1 ----- II--Order---- --1 (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 B7 V. Description of the invention (Y > 1)% methanamine, 0.2% SDS, 5x Denfardt ' The solution, 0.1 mg / ml salmon sperm DNA, and 6 x standard citrate (SSC) pre-hybridization buffer were pre-hybridized at 42 ° C for 1 hour. The 1.0 仟 base lambda phage DNA. Fragment prepared in Example 1- (1) was labeled with Cy5TM according to the instruction manual by using Label IT® Cy5TM labeling kit (Minis). The labeled nucleic acids were purified by ethanol precipitation. Dilute this Cy5TM-labeled nucleic acid probe with pre-hybridization buffer to a final concentration of 20, 2 and 0.2 ng / ml, heat at 95 ° C for 2 minutes, and cool to room temperature. The insoluble material was removed by centrifugation. Approximately 5 microliters of the hybridization solution was dropped on the nucleic acid array. Carefully cover a cover slip to avoid air bubbles, and incubate in a constant humidity device at 42 ° C for 20 hours. The coverslips were removed in 2x SSC solution at room temperature, washed with 0.2% SDS containing 2x SSC, washed twice at 65 ° C for 5 minutes, and washed at 55 ° C for 30 minutes. Then, it was washed at 0.05 x SSC for 10 minutes at room temperature, and then removed by centrifugation at a speed of about 1,000 rpm, and then air-dried. After hybridization, the nucleic acid array was read by using a GMS 418 array scanner (GMS) with a divergence wavelength of 635 nm and an excitation wavelength of 660 nm. The detected signal strength is analyzed by the image analysis software ImaGene (Biodiscovery). The results obtained are summarized in Table 1. 15 This paper size applies to China National Standard (CNS) A4 (21〇X 297 mm) ϋ · ϋ I —Bi I ni ^ i · mmmmm ϋ I. n _ϋ · —Bi ϋ I _ tl (Please read first Note on the back, please fill in this page again) 1224619 A7 B7 Printed by the Consumers' Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention Table 1 Probe-fixed DNA fragments are used for DNA-immobilized substrate concentration activated ester-coated PLL coated (neck / ml) (base pairs) —__ glass slide glass 0.2 1000 1484 889 300 988 856 2 1000 6640 2702 300 2635 1077 20 1000 27383 14310 300 7099 3824 As shown in Table 1, At all probe concentrations, the signal of each size of immobilized DNA on the activated ester-coated glass slide was greater than the signal produced on the PLL-coated glass slide. That is, in the detection of the target PCR amplified nucleic acid fragments, it was shown that DNA wafers prepared using activated ester-coated glass slides can be prepared using more commonly used (PLL) -coated glass slides. DNA chip, provides higher signal. Example (Explanation) 3 (1) Construction of a DNA wafer with an oligonucleotide-DNA array Two 24 nucleotide oligonucleotides were prepared on a DNA synthesizer; in the sequence listing, oligonucleotides The acid is shown as a sequence number: 6 'One of the oligonucleotides has an alkylamino group of 6 (PE Biosystems) with a chain length of 6' modified at the 5, end (abbreviated as NH2-F 〇lig〇) 'and another Oligonucleotide is modified at the 5 'end to include a hydroxyl hydrazone (abbreviated as 0H_F 01ig〇16) This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I ^ 1 ^ 1 1 > 1. ^ 1 ^ 1 ϋ ϋ n ϋ I ϋ ϋ I ϋ I 1 ej -ϋ I ϋ ϋ ϋ ϋ ϋ I (Please read the notes on the back before filling out this page) 1224619 A7 B7 V. Description of the invention (V <)). Two other oligonucleotides with a length of 24 nucleotides were also synthesized; one of the oligonucleotides had a sequence complementary to the first oligonucleotide showing the sequence number: 7 and had Cy3TM at the 5 'end ( Cy3-R 01igo); and another oligonucleotide has a sequence unrelated to the sequence number shown: 14 and has an alkylamine linker (NH2-N01igo) at the 5 'end. OH-F Oligo, NH2_F Oligo, and NH2-N Oligo were dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 5, 50, and 500 // M, and then arranged in Example 1- (4) The method is activated on an activated ester-coated glass slide. At that time, 7 spots were configured for each DNA sequence. After spotting, the slide was rinsed and dried as described in Example 2, and finally processed into an oligonucleotide wafer. As a control group, the same oligonucleotide array was prepared on a glutaraldehyde-treated slide (as described in Example 1- (2)). (2) Hybridization reaction of oligonucleotide wafer
將製作在每個載片上的寡核苷酸晶片，以分開製備的 Cy3-R Oligo爲探針而雜交，並觀察所得的螢光訊號。簡言 之，將大約 5 微升含有 1 mM Cy3-R Oligo、〇.2%SDS、5x Denfardt’溶液、0·1毫克/毫升鮭魚精子DNA以及6x SSC 的雜交溶液，加到寡核苷酸陣列。小心覆蓋一層蓋玻片， 以避免氣泡形成，並將其在溼度恆定裝置中，以42°C培養 20小時。將蓋玻片在室溫下，在2x SSC溶液中移除，並 以2x SSC在室溫下淸洗10分鐘兩次。然後，藉由在大約 1，000轉/分鐘的低轉速離心而移除溼氣，然後風乾。雜交 之後，使用具有635 nm發散波長以及660 nm激發波長的 17 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 一 I Mmmmm i mmmmw mmew ϋ J I i I emmmm i t§ l^i —Bi I ·ϋ I i (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（A) GMS 418陣列掃描儀，而測量寡-DNA晶片上的總螢光。 偵測的訊號強度藉由影像分析軟體IniaGene而分析。所得 到的結果摘述於表2中。 表2 寡核苷酸點陣溶液 之濃度UM) 寡-DNA 活化的酯類塗覆的 載片玻璃 f 戊二醛處理的 載片玻璃 5 OH-F Oligo 19.8 0.0 NH2-F Oligo 5516.7 0.9 NH2-N Oligo 0.0 0.0 50 OH-F Oligo 2067.0 0.0 NHrF Oligo 12104.1 0.0 NH2-N Oligo 0.0 0.0 500 OH-F Oligo 4385.8 1113.5 NH2-F Oligo 14828.7 201.9 NH2-N Oligo 0.0 8.5 如表2所示，在活化的酯類塗覆的玻璃載片上，在5’ 端具有胺基之寡核苷酸點陣，較不具胺基之寡核苷酸點陣 顯示較強的訊號。這顯示在載片玻璃上之活化的酯類基團 ，已與寡核苷酸的胺基反應。在所有的寡核苷酸中，在活 化的酯類塗覆的玻璃載片上之點陣，其訊號均大於戊二醛 處理的玻璃載片上所產生的訊號。 實施例（說明）4 聚丙烯酸的立體效應 從核酸陣列所得的雜交訊號，在其上已點上不同長度 18 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)The oligonucleotide wafer prepared on each slide was hybridized with Cy3-R Oligo prepared separately as a probe, and the resulting fluorescence signal was observed. Briefly, approximately 5 μl of a hybridization solution containing 1 mM Cy3-R Oligo, 0.2% SDS, 5x Denfardt 'solution, 0.1 mg / ml salmon sperm DNA, and 6x SSC was added to the oligonucleotide Array. Carefully cover a cover slip to prevent air bubbles from forming, and incubate in a constant humidity device at 42 ° C for 20 hours. The coverslips were removed in 2x SSC solution at room temperature and rinsed twice with 2x SSC at room temperature for 10 minutes. Then, the moisture was removed by centrifugation at a low speed of about 1,000 rpm, and then air-dried. After hybridization, 17 paper sizes with a divergent wavelength of 635 nm and an excitation wavelength of 660 nm were applied to the Chinese National Standard (CNS) A4 (210 X 297 mm)-I Mmmmm i mmmmw mmew ϋ JI i I emmmm it§ l ^ i —Bi I · ϋ I i (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 B7 V. Description of the invention (A) GMS 418 array scanner, which measures -Total fluorescence on DNA chip. The detected signal strength is analyzed by the image analysis software IniaGene. The results obtained are summarized in Table 2. Table 2 Concentrations of the oligonucleotide lattice solution UM) Oligo-DNA activated ester-coated slide glass f Glutaraldehyde-treated slide glass 5 OH-F Oligo 19.8 0.0 NH2-F Oligo 5516.7 0.9 NH2- N Oligo 0.0 0.0 50 OH-F Oligo 2067.0 0.0 NHrF Oligo 12104.1 0.0 NH2-N Oligo 0.0 0.0 500 OH-F Oligo 4385.8 1113.5 NH2-F Oligo 14828.7 201.9 NH2-N Oligo 0.0 8.5 As shown in Table 2, in the activated ester On a coated glass slide, an oligonucleotide dot array with an amine group at the 5 'end shows a stronger signal than an oligonucleotide dot array without an amine group. This shows that the activated ester group on the slide glass has reacted with the amine group of the oligonucleotide. In all of the oligonucleotides, the dots on the activated ester-coated glass slides produced greater signals than those produced on glutaraldehyde-treated glass slides. Example (Explanation) 4 Three-dimensional effect of polyacrylic acid The hybrid signals obtained from nucleic acid arrays have been marked with different lengths 18 This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) (please first (Read the notes on the back and fill out this page)
經濟部智慧財產局員工消費合作社印製 1224619 A7 B7 五、發明說明（1) 之螢光標示的核酸’是以每個固定的核酸長度之訊號而計 算。人類鐵傳遞蛋白受體（簡寫爲TFR)的CDNA片段， 是藉由下列方法而製備。根據正常的方法而從人類細胞株 K562 (Dainippon Pharmaceutical)中萃取 mRNA ’ 並製備 TFR的cDNA。已上述的cDNA作爲模板，使用在序列表 中TFR (基因資料庫編號：X〇l〇6〇)專一性引子，引子S7 及引子AS7，分別爲序列編號：8及序列編號：9，製備 4,738鹼基對的PCR產物。藉由將上述的PCR片段與 pT7Blue載體（Novagen)連接，而得到重組的質體 pT7TFR。藉由PCR，分別使用在5’端標示鹼性蕊香紅X ( PE Biosystems ;簡寫爲ROX )的引子對中的一^個以及 pT7TFR作爲引子及模板，而製備不同長度之螢光標示的 PCR片段，也就是，使用以ROX標示的S7 (序列編號：8 )及AS4 (序列編號：10)之引子對、AS3 (序列編號： 11)、AS2 (序列編號：12)或AS1 (序列編號：13)， 而進行PCR。結果，分別製備了 1.0、0.5、0.2及0.1仟鹼 基的片段。 純化之後，將每個DNA片段溶解在20 mM MOPS緩 衝液（pH 7.5)中，並使用Amina Label IT®試劑而胺化， 也就是，將Amino Label IT®試劑溶解在二甲基亞硼，以 1/5的比例（重量/重量）加到純化的DNA溶液中，並在 37°C培養1小時。每個胺化的DNA藉由酒精沈澱法而純化 〇Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 B7 V. Description of the Invention (1) The fluorescently labeled nucleic acid ’is calculated for each fixed nucleic acid length signal. The CDNA fragment of human transferrin receptor (abbreviated as TFR) was prepared by the following method. According to a normal method, mRNA 'was extracted from a human cell line K562 (Dainippon Pharmaceutical), and TFR cDNA was prepared. The above-mentioned cDNA was used as a template, and TFR (gene database number: X〇106) specific primers in the sequence table, primer S7 and primer AS7, were sequence number: 8 and sequence number: 9, respectively. Preparation 4,738 Base pair PCR product. Recombinant pT7TFR was obtained by ligating the aforementioned PCR fragment with a pT7Blue vector (Novagen). By PCR, one ^ of primer pairs labeled with basic rue red X (PE Biosystems; ROX for short) at the 5 'end and pT7TFR were used as primers and templates to prepare fluorescently labeled PCRs of different lengths. Fragment, that is, using primer pairs S7 (sequence number: 8) and AS4 (sequence number: 10), AS3 (sequence number: 11), AS2 (sequence number: 12), or AS1 (sequence number: 13), and perform PCR. As a result, 1.0, 0.5, 0.2, and 0.1 sulfonyl base fragments were prepared, respectively. After purification, each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) and aminated with Amina Label IT® reagent, that is, Amino Label IT® reagent was dissolved in dimethylboron to A 1/5 ratio (weight / weight) was added to the purified DNA solution and incubated at 37 ° C for 1 hour. Each aminated DNA was purified by alcohol precipitation.
Amino Label IT試劑是如專利申請案W098/52961中 19 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I ϋ ϋ 1 ·ϋ ϋ ϋ I ·1 ^1 I ϋ I · 言 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224619 A7 ______ B7 五、發明說明（d) 所說明的方法而製備，將氟基取代爲胺基。 將胺化的TFR片段溶解在20 mM MOPS緩衝液（pH 7·5)中，至最終濃度爲0.5毫克/毫升，然後藉由實施例ΙΟ) 之方法 ，將每個序列的 7 個點陣 ，點在如實施例 1-(4)所活化之活化的酯類所塗覆的載片玻璃上。點上去之 後，如實施例3中所說明的方法淸洗及乾燥。作爲對照組 ，如實施例1- ( 1 )，在MAS塗覆的載片玻璃上製備相同 的陣列，並以丁二酸酐處理或不處理，以封阻在固定之後 的游離胺基。 在每個載片坡璃上製作的DNA晶片，是以Cy5標示 的DNA探針而雜交，其具有不同於ROX的螢光波長，並 且觀察它們的訊號。也就是，將上述製備的1.0仟鹼基 TFR片段，藉由使用Cy5 Label IT®套組試劑（Mirus)， 根據使用手冊而以Cy5TM標示。 將大約5微升的雜交溶液，其含有20奈克/毫升以 Cy5 標示的 DNA 探針、0.2%SDS、5x Denfardt 溶液、0.1 毫克/毫升鮭魚精子DNA以及6x SSC，加到在晶片上DNA 固定的區域。將其覆蓋一層蓋玻片，並在溼度恆定裝置中 ，以65°C培養20小時。將蓋玻片在室溫下，在2x SSC溶 液中移除，以含有2x SSC的0_2%SDS，在65°C淸洗5分 鐘兩次，以及在55°C淸洗30分鐘。然後，將其在室溫下 ，以0.05x SSC淸洗10分鐘，藉由在大約1，000轉/分鐘的 低轉速離心而移除溼氣，然後風乾。 在雜交之後，在陣列DNA末端標示的ROX以及在 20 1 ϋ ·ϋ 1 1 · 1·— ϋ ϋ Βϋ I I · 言 (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1224619 A7B7 五、發明說明（i| ) DNA晶片上的雜交探針所標示的Cy5，其螢光訊號是使用 GMS 418陣列掃描儀而測量。所得的影像藉由影像分析軟 體ImaGene而分析，並將螢光訊號轉變成數値資料。將 Cy5的螢光強度數値，除以每個DNA長度中ROX的螢光 .強度數値，將其視爲訊號強度/ DNA。結果摘述於表3中 。因爲有高的背景値，從未封阻游離胺基之MAS塗覆的載 片玻璃上所製作的DNA晶片中，要測量其訊號是不可能的。 表3 DNA大小 (鹼基對） 訊號強度/DNA (A)/(B) 活化的酯類塗覆的 載片玻璃（A) MAS塗覆的 載片玻璃（B) 1000 26.77 9.09 2.9 500 9.61 4.17 2.3 200 0.46 0.43 1.1 100 0.30 0.18 1.7 如表3 所示，在所有的 DNA片段長度中 ，在活化的 酯類塗覆的載片玻璃上，每DNA的點陣訊號強度，均大於 在MAS塗覆的載片玻璃上，每DNA的點陣訊號強度。這 個結果顯示，活化的酯類塗覆的載片玻璃可提供更有效的 雜交反應。由共價貼附的聚陰離子所產生的立體點陣，可 對於貼附的標的核酸提供更大的通道° 實施例（說明）5 將1.0仟鹼基及0.3仟鹼基兩種大小，在其末端具有 (請先閱讀背面之注意事項再填寫本頁) tr---------^91. 經濟部智慧財產局員工消費合作社印製 21 1224619 A7 ______B7_____ 五、發明說明（?/〇) 胺基的λ噬菌體DNA片段，藉由與實施例2- ( 1)中相同 的方法而製備。DNA的一部份，藉由使用如實施例4中所 說明之Amino Label IT®試劑而胺化。將每個DNA片段溶 解在20 mM MOPS緩衝液（pH 7.5 )中，至最終濃度爲0.5 毫克/毫升，並以7個不同的位置，點在活化的酯類所塗覆 的載片玻璃以及MAS塗覆的載片上。 在每個載片玻璃上製作的核酸陣列，是以螢光Cy3標 示的DNA探針而雜交，並分析所得的訊號。也就是，將上 述製備的1.0仟鹼基λ噬菌體片段，藉由使用Label IT® Cy3標示套組試劑（Minis)，根據使用手冊而以Cy3標示 。使用Cy3標示的DNA探針，如實施例4中所說明的方 法，進行雜交反應及淸洗。 在雜交之後，使用具有532 nm發散波長以及570 nm 激發波長的GMS 418陣列掃描儀，測量DNA晶片的螢光 (也就是，訊號強度）。分析所得的影像，並將訊號強度 的數値，藉由使用影像分析軟體ImaGene而轉變成數値資 料。藉由在MAS塗覆的載片玻璃上之點陣，而計算相對的 螢光強度爲1〇〇。所得的結果摘述於表4中。 I ·1 1 1 ϋ ϋ βΜΒ «I > iBi ϋ ϋ ϋ n ϋ ϋ I . 言 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 表4 標的DNA 訊號的相對強度 大小 藉由 Label IT MAS塗覆的 活化的酯類塗覆的 (鹼基對) 之胺基修飾 載片玻璃 載片玻璃 1000 - 100 183 1000 + 100 276 300 — 100 945 300 + 100 2492 22 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1224619 A7 ____B7____ 五、發明說明（4) 如表4所示，在所有的DNA片段點陣中，由活化的 酯類塗覆的載片玻璃所得之訊號強度，均高於從MAS塗覆 的載片玻璃上所得之訊號強度。特別地，300個鹼基對的 短DNA片段之訊號，是更高於MAS載片上所得之訊號。 當點在活化的酯類塗覆的載片玻璃上時，具有胺基修 飾之標的物，較沒有胺基修飾者產生更強的訊號。這證明 以Amino Label IT®的胺基修飾，可有效地提供標的物對於 活化的酯類塗覆的玻璃載片之增加的貼附。 上述的實施例僅是作爲本發明原理的舉例說明。此外 ，由於熟悉於此技藝者可容易地做更種的潤飾及更動，因 此，並無意將本發明限制在上述所顯示及說明的精確建構 及操作中。因此，適當的潤飾以及等價物也是在本發明的 範疇之內。 --------------------噑. (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 23 申讀曰期 η.Ί、。 案 號 類 別 C/nM 1 In 今知 (以上各欄由本局填註） •d士二 口 f|專利説明書1224619 _、發明 參 新雙名稱 中 文 用於核酸固定化之由塗覆有聚丙烯酸之無孔性玻璃 載片所組成之基材 英 文 Substrate Consisting of Non-porous Slide Glass Coated with Polyacrylic Acid for Nucleic Acid Immobilization 姓_ 名 ⑴強A.沃爾夫 ⑷瑪莉-安瓦特 (2) 詹姆士 E.哈格斯托姆 （5){呆羅M.史拉坦 (3) 維拉地米爾G.巴得克 二、發明 創作人 國 籍 美 國 住、居所 (1庚國·纖槺新州53705麥狄遜，大學灣路1122號 (2庚國·廊牖新州535ffi密得坦，伯格莫特路4700號 (3庚國·靡斤藤斤州53705麥挑1,67A公寓，北画璐406號 (燠國·廊斤麵州53Ή9麥麵，C公寓，洛得克里夫路7617號 (5庚國·纖慷新州53Ή1麥狄遜凱丁路4306號 姓 名 (名稱） 寶生命工學股份有限公司 國 籍 曰 本 三、申請人 住、居所 (事務所） 曰本滋賀縣大津市瀨田三丁目4番1號 / 代表人 姓 名 加藤郁之進 Γ7— _厂…---- 裝 訂 線Amino Label IT reagent is as in patent application W098 / 52961 19 This paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) I ϋ ϋ 1 · ϋ ϋ ϋ I · 1 ^ 1 I ϋ I · (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224619 A7 ______ B7 V. Prepared by the method described in (d) of the invention, replacing the fluoro group with an amine group. The aminated TFR fragment was dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 0.5 mg / ml, and then by the method of Example 10), 7 dots of each sequence were obtained. Spot on glass slides coated with activated esters activated as in Example 1- (4). After spotting, it was rinsed and dried as described in Example 3. As a control group, as in Example 1- (1), the same array was prepared on a MAS-coated slide glass, and treated with or without succinic anhydride to block free amine groups after immobilization. DNA wafers made on each slide were hybridized with DNA probes labeled with Cy5, which had fluorescence wavelengths different from ROX, and their signals were observed. That is, the 1.0-base TFR fragment prepared as described above was labeled with Cy5TM according to the instruction manual by using the Cy5 Label IT® kit reagent (Mirus). Add approximately 5 μl of hybridization solution containing 20 ng / ml of DNA probe labeled Cy5, 0.2% SDS, 5x Denfardt solution, 0.1 mg / ml salmon sperm DNA, and 6x SSC to DNA fixation on the wafer Area. Cover it with a cover slip and incubate at 65 ° C for 20 hours in a constant humidity device. The coverslips were removed in 2x SSC solution at room temperature, rinsed twice at 65 ° C for 5 minutes with 0_2% SDS containing 2x SSC, and rinsed at 55 ° C for 30 minutes. Then, it was rinsed at 0.05 x SSC for 10 minutes at room temperature, and the moisture was removed by centrifugation at a low speed of about 1,000 rpm, and then air-dried. After hybridization, the ROX marked at the end of the array DNA and 20 1 ϋ · ϋ 1 1 · 1 · — ϋ ϋ ϋ ϋ ϋ ϋ 言 (Please read the precautions on the back before filling out this page) This paper size applies to Chinese national standards (CNS) A4 specification (210 X 297 mm) 1224619 A7B7 5. Description of the invention (i |) Cy5 marked by the hybridization probe on the DNA wafer, the fluorescence signal is measured using a GMS 418 array scanner. The obtained image is analyzed by the image analysis software ImaGene, and the fluorescent signal is converted into digital data. Divide the fluorescence intensity 値 of Cy5 by the fluorescence intensity 强度 of ROX in each DNA length, and consider it as signal intensity / DNA. The results are summarized in Table 3. Due to the high background radon, it is impossible to measure the signal in DNA wafers made from MAS-coated slide glass that has never blocked free amine groups. Table 3 DNA size (base pairs) Signal strength / DNA (A) / (B) Activated ester-coated slide glass (A) MAS-coated slide glass (B) 1000 26.77 9.09 2.9 500 9.61 4.17 2.3 200 0.46 0.43 1.1 100 0.30 0.18 1.7 As shown in Table 3, the dot matrix signal strength of each DNA on activated ester-coated slide glass is greater than that of MAS coating in all DNA fragment lengths The intensity of the dot matrix signal per DNA on the slide glass. This result shows that activated ester-coated slide glass can provide a more efficient hybridization reaction. The three-dimensional lattice produced by the covalently attached polyanions can provide larger channels for the attached target nucleic acid. Example (Explanation) 5 Two sizes of 1.0 仟 base and 0.3 仟 base are provided in the The end has (Please read the notes on the back before filling this page) tr --------- ^ 91. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 21 1224619 A7 ______B7_____ V. Description of the invention (? / 〇 Amino λ phage DNA fragment was prepared by the same method as in Example 2- (1). A portion of the DNA was aminated by using the Amino Label IT® reagent as described in Example 4. Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 0.5 mg / ml, and spotted glass and MAS coated with activated esters were spotted at 7 different positions Coated slides. The nucleic acid array prepared on each slide glass was hybridized with a DNA probe labeled with fluorescent Cy3, and the resulting signal was analyzed. That is, the 1.0 仟 base lambda phage fragment prepared as described above is labeled with Cy3 according to the instruction manual by using Label IT® Cy3 labeling kit (Minis). A Cy3 labeled DNA probe was used for the hybridization reaction and washing as described in Example 4. After hybridization, the fluorescence (ie, signal intensity) of the DNA wafer was measured using a GMS 418 array scanner with a 532 nm divergence wavelength and an 570 nm excitation wavelength. The obtained image is analyzed, and the data of signal strength is converted into data by using image analysis software ImaGene. The relative fluorescence intensity was calculated from the lattice on the MAS-coated slide glass to 100. The results obtained are summarized in Table 4. I · 1 1 1 ϋ Μ βΜΒ «I > iBi ϋ ϋ ϋ n ϋ ϋ I. Words (please read the precautions on the back before filling out this page) The Intellectual Property Bureau of the Ministry of Economic Affairs's Consumer Cooperatives printed the DNA signal of the subject 4 The relative strength of the substrate was modified by the use of Label IT MAS activated ester-coated (base pair) amine modified slide glass slide glass 1000-100 183 1000 + 100 276 300 — 100 945 300 + 100 2492 22 This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) 1224619 A7 ____B7____ 5. Description of the invention (4) As shown in Table 4, in all the DNA fragment lattices, coated with activated esters The signal strength obtained from the coated slide glass is higher than the signal strength obtained from the MAS-coated slide glass. In particular, the signal of a short DNA fragment of 300 base pairs is higher than that obtained on a MAS slide. When spotted on an activated ester-coated slide glass, the target with an amine-based modification produces a stronger signal than the one without the amine-based modification. This proves that the amine modification with Amino Label IT® is effective in providing increased adhesion of the target to activated ester-coated glass slides. The above-mentioned embodiments are merely illustrative for the principle of the present invention. In addition, since those skilled in the art can easily make more modifications and changes, it is not intended to limit the present invention to the precise construction and operation shown and described above. Therefore, proper retouching and equivalents are also within the scope of the present invention. -------------------- 噑. (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs η.Ί ,. Case No. Category C / nM 1 In Now Known (The above columns are filled by this Office) • d 士 二 口 f | Patent Specification 1224619 _ 、 Invention and New Double Name Chinese for nucleic acid immobilization coated with polyacrylic acid Substrate Consisting of Non-porous Slide Glass Coated with Polyacrylic Acid for Nucleic Acid Immobilization Substitute_ First Name Qiang Qiang A. Wolff Mary-Anwart (2) James E. Hagerstom (5) {Daluo M. Slatan (3) Veramir G. Bardeck II. Inventor and Creator Nationality U.S. Residence and Residence 53705 Madison, No. 1122 University Bay Road (2 Geng State · Langfang NSW 535ffi Middletown, 4700 Bergmoth Road (3 Geng State · Mi Jin Teng Jinzhou 53705 Mai Tiao 1, 67A Apartment, North Hualu No. 406 (Languo · Langjin Mianzhou 53Ή9 Wheat Noodles, Apartment C, No. 7617 Lot Cliff Road (5 Geng Guo · Xianggan NSW 53Ή1 Madison Kaiding Road 4306) Name (Name) Bao Life Engineering Co., Ltd. Nationality Japanese Version III. Applicant's Residence and Residence (Office) Japanese Version Otsu, Shiga Prefecture Tian Fan Sanchome 4 1 / representative Name Yu Kato Γ7- _ into the plant ... ---- stapling line