1287581 九、發明說明： 【發明所屬之技術領域】 本I:月疋有關於—種微量細胞核酸檢測晶片之方 >:尤指-種可達到成本低、操作易、功效高的基因晶 2 ’讓生物基因晶片的功能與應用能有效 X JL只日及將基因晶片實際應用於各相關領域中。 【先前技術】 按二生物晶片係為—種微型裝置，通常以石夕晶片、 玻璃或南分子為基質，计 ^ . 、 亚以彳政小化技術整合生物有機分 生物，fU酸或蛋Μ)為生化探針，用來檢測或分析 行八生物晶片的體積小、反應快速’並且能夠平 产.1里生物貝訊’因此，可適用於生化處理、分析、 U、，開發及環境監測等用途上。 片，1I2:發展的最完整並最受矚目的就是基因晶 乃其主要特點如下： (1)同時谁并士旦 據不同之研究目的Γ:同基因之檢測:研究人員可根 # 勺，込擇不同的核苷酸片段，並將該核 甘酉欠片段固定在曰y L ^ „ 仕日日片上，使其排列整齊成陣；一般而言， 檢二大此來研究細胞内基因的表現’達到快速 豆一二 功效，同時也改變了以往之研究人員終 研:種基因的研究方式。 斤而生物性材料：所需之檢體樣本，生化探對 1287581 及標的物等皆減少，與過去傳統之 之檢體量減少達2〇倍以上之多。 口”沾墨點法」所需 援，^ ^動化分析：具有各種生物資訊相關軟體支 效;且不但可以達達到完全自動化之功 功能，更二 量、差異基因搜尋兩大基本 刀月匕’更月b父互運用而料吐ψ夕 十 於生物醫學上φ ^ / 夕方面之應用，不僅應用 軍事用、： 更廣佈食品檢驗、化學合成、新藥研發、 早爭用逯...等多種領域上。 類：然而至目前為止，其基因晶片的發展主要分為兩大 产理t日 ㈣晶片：—般玻璃晶片經由特殊方5 ί 1广以將核苷酸片段固定在晶片上，排列整齊成p ’衣備為所需之基因晶片；此種晶片無法普 種相關領域’主要原因如下： 、c 高，大 玻璃處理技術之門檻高，且成本亦相對的提 " 匕並非一般貫驗室或檢測單位所能負擔。 (2)玻璃載體晶片所使用之螢光呈色暨雜交反應· =檢體（核糖核酸）品質要求高、反應步驟繁輕技 1難榮光標不物（cy3及cy5)成本高且使用不易。 定j 3)結果螢光呈色之結果分析，所需之掃描器為特 疋之知描器，其成本高昂絕非一般實驗單位所能負擔。 龍二）·尼龍膜晶片··將所需之核苷酸片段固定在尼 月果上，製備晶片。相較於玻璃晶片；尼龍模晶片製備 1287581 牛易μ戶、驗技術門檻低，且其所使用之化學呈色法實驗 :驟簡易、忒劑成本低，但是，此種方法所需之檢體（核 、唐核里較大，化學呈色結果分析無自動化之分析， =導致嚴重影響實驗錄度及精確性；因此，該尼龍 模晶片反而漸漸被玻離晶片所取代。 ^除此之外，無論玻璃晶片或尼龍模晶片製備時，均 =^動化點陣設備，而該自動化點陣設備之價格亦相當 =叩，且儀器操作不易，就現況而言，即使能操作晶片 貫驗：研究室也都無法獨立製備所需之晶片，而須轉由 ^特定生技公司代而執行之；故，上述„般傳統之基因 曰曰片製備方法並無法符合實際使用時之所需。 1287581 【發明内容】 因此，本發明之主要目的係在於，可藉由本發明之 方法達到成本低、操作易、功效高的基因晶片操作技術 平台，讓生物基因晶片的功能與應用能有效發揮。 本發明之另一目的係在於，可藉由本發明之方法達 到確實普及將基因晶片實際應用於各相關領域中。 為達上述之目的，本發明係一種微量細胞核酸檢測 晶片之方法，其包括下列步驟： a. 選定一尼龍模晶片，利用一手動打點機，將所需之基 因核苷酸片段整齊排列點陣於晶片上； b. 手動打點後自然烘乾，再以一核酸快速固著器 (cross_linker)，將核苷酸片段固著於尼龍模上，即 完成晶片製備； c. 收集一般血液作為檢體； d. 萃取上述血液中之核糖核酸並進行線性放大； e_反轉錄試驗合成足量所需之互補去氧核糖核酸 (cDNA)並加以標誌，使其形成一作為探針之標誌 物； f. 將上述之晶片與標誌物進行探針標誌反應、雜交反應 (Hybridization)以及雜交後反應（Post-Hybridization) 處理； g. 將上述雜交後反應之晶片與標誌物進行化學呈色反 應； h_將上述化學呈色反應後之影像結果進行自動化分析。 1287581 ί 如是，藉由上述之步驟 功效高的基因晶片操作技術平么達」成本低、操作易、 能與應用能有效發揮，並確“ W的功 於各相關領域中。 曰：土因晶片實際應用 【實施方式j 請參閱『第1、2、？网 ^ 古诒+立闰n d圖』所示，係本發明之流卷 方塊不思圖、手動打點應夕+ U铖之立體外觀示意圖、手動打$ 機之立體分解示意圖。如罔邮-丄 卞勒打點 、 〜口如圖所不··本發明係一種微量細 胞核酸檢測晶片之方法，其步驟包括·· a·選定—尼龍模晶片，利用-手動打點機i，將每— 基因核魏諸以⑽n丨之大小、15_間距點陣排列 於晶片上，並將所需之基因核㈣片段以二次水泡製成 200μΜ以100n丨之大小整齊排列點陣於晶片上，而該手 動打點機1 (如第2、3圖所示）係包括有—底座丄丄、 口又置於底座1 1上之晶片放置層1 2、一設置於晶片 放置層1 2上之晶片固定層1 3、及一設置於晶片固定 層1 3上之打點層1 4所構成，該晶片放置層丄2及打 點層1 4之一侧係以一夾層樞紐1 5加以連接，藉以使 °亥打點層1 4可作開啟之動作，且該打點層1 4之適當 處係設置有一啟動手把1 4 1，並於該打點層1 4之正 中央處係設置有多數個孔洞1 4 2，而該打點層1 4之 正中央處係選取4·5x4·5 cm的區塊，並以3mm的孔距 製備出196個（行：14 ;列：14 )内徑1 2 mm的孔洞1 1287581 4 2 ’做為之後微量吸取管插人處（即打點處）； 而該手動打點機之使用方法如下： 上拉料動打關1之啟動手把141打開打 …占層14，將晶片放置於晶片放置層i 2。 —2·蓋上打點層14，晶片固定層13自動將晶片固 疋0 3_以微量吸量管吸取泡製好之寡核糖核酸，直接深入 打點層1 4中央之孔洞1 4 2，打人寡核糖核酸即可。 b.於手動打點後自然烘乾，再以一核酸快速固著器 日(cr〇ss-lmke「），將核:酸片段固著於尼龍模上，即完成 製備’該核酸快速固著器係以12QQ焦耳之高能量進 仃父叉結合（crosslink)，而將核苷酸片段固著於尼龍模 上； C·收集一般血液作為檢體，而於收集檢體時僅需1c c 的血液檢體即可進行反應得取40單位的CDNA (互補去 氧核糖核酸），且每次晶片實驗所需之cDNA僅需4單位； d_萃取上述血液中之核糖核酸並進行線性放大； e·反轉錄試驗合成足量所需之cdNA並加以標誌，使 其形成一作為探針之標誌物； f·將上述之晶片與標諸物進行探針標誌反應、雜交反 應（Hybridization)以及雜交後反應（p〇s 卜 Hybr1dizat•丨 〇n) 處理，該探針標誌反應時間及雜交反應時間係為24小時； g·將上述雜交後反應之晶片與標誌物進行化學呈色 1287581 反應，該雜交後反應處理時，其偵測（Detection)的時 間為30分鐘，可使該雜交後反應之呈色反應加速，使背 景顏色不至於因過久的成色而造成過度呈色，並且可降 低反應日夺之誤測（False Positive reaction )，且該雜交之 後及固定步驟前的薄膜（membrane)清洗，係可使用自 行調配之清洗試劑（Washing buffer)，而該呈色前的薄 膜（Membrane )處理係可使用自行調配之偵測試劑 (Detection buffer)； h.將上述化學呈色反應後之影像結果進行自動化分 析，而該呈色反應後之影像結果係可利用一般密度 (Density)分析軟體自動分析。 請參閱第4圖所示，首先我們先以生物統計軟體 (Statistical Package for the Social Sciences Ver. 10.0)分析實驗結果，從所得的接收者操作特徵曲線 (Receiver Operating Characteristics curve)得知，在判 定此次所用每片晶片上22個基因與正常基因表現值比較 後，若有11個或11個以上基因有兩倍以上的過度表現， 即為正反應（positive，十），當11個以下時即判定此晶 片為負反應（negative，一），如此會得到最合理的統計， 其靈敏度和特異性也會落在最適當點，因此以此數據作 為判讀的標準。 請參閱第5圖所示，為了確認這個方法的可行性及 其敏感度，我們特地進行在玻璃器内的實驗，將事先前 1287581 建立好的K-ras致癌基因穩定轉染之人類腎上腺皮質細 胞株（K-ras mutant stably transfected adrenocortical cells，K-ras MSTAC)置入正常血液中後，與本發明之 檢測晶片反應進行後進行檢測晶片分析，該檢測晶片係 使用正控制（ + )，且該正控制係採用β—肌動蛋白1 6。 其結果，當晶片上有22個基因，而1c.c.的血液中含有 20個K-ras MSTAC細胞時，可得到有16個基因過度表 現，此晶片判讀為正反應，也就是說有72.72%的基因表 現為正（positive，+ )，大於50% ;而當1c_c含有5個 K-ras MSTAC細月包時’貝U有12個基因過度表現’此晶片 判讀為正反應，也就是有54.55%的基因表現為正，亦大 於50% ;但是當細胞數減為2個時，僅剩3個基因過度 表現，則此晶片判讀為負反應，也就是只有13.64%的表 現為正，小於50% ;當細胞數僅1個時，過度表現之基 因更少於3個而小於50%，故結果仍判讀為負反應。因 此本發明檢測晶片確實可行且只要每一 c.c血液細胞數 大於5，則我們的檢測晶片即可精確的偵測到血液中 K-ras基因活化情形。 請參閱『第6圖及表一』所示，係本發明利用微量 細胞核酸檢測晶片技術平台檢測各種癌症病人血液中細 胞與特定基因群之反應表現示意圖、本發明利用即時聚 合酶鏈鎖反應（Real-Time PCR)確認微量細胞核酸檢 測晶片技術平台檢測結果。如圖所示：其中該第4圖係 12 1287581 ^ = :2癌症病人血液的核糖核酸與本發明的診斷 日日片反應的結果；而嗜矣一 因為即時聚合酶鏈錯“3為即時聚合酶鏈鎖反應’ __ ffl —、’、貞反應是另一種檢測RNA表現的技 :用試相同基因在本發明所使用之診斷晶片及 否 ^K ° _鏈鎖反應所測得知基因放大表現情形是 放大的絲—可知，本發9㈣實在晶片上所測得 ^大的基因在即時聚合酶鏈鎖反應也—樣有放大的情1287581 IX. Description of the invention: [Technical field to which the invention pertains] This I: There is a method for detecting a wafer of a small amount of cellular nucleic acid in the moon &; in particular, a gene crystal 2 which can achieve low cost, easy operation, and high efficacy 'Let the function and application of bio-gene wafers be effective X JL only and the gene chip is actually applied in various related fields. [Prior Art] According to the second bio-chip system, it is a micro-device, usually based on the Shixi wafer, glass or southern molecules, and the integration of bio-organisms, fU acid or egg tarts. As a biochemical probe, it is used to detect or analyze the volume of the eight biochips, and the reaction is fast 'and can produce a flat product. 1 bio-bein' is therefore suitable for biochemical treatment, analysis, U, development and environmental monitoring. For other purposes. Film, 1I2: The most complete and most noticeable development is GeneCell. Its main features are as follows: (1) Whoever is based on different research purposes: Detection of the same gene: Researcher can root # spoon, 込Select different nucleotide fragments, and fix the nuclear glycoside fragment on 曰y L ^ „ Shiri Japanese tablets, so that they are arranged in a neat array; in general, the second big test to study the performance of genes in cells 'To achieve the effect of fast bean one or two, but also changed the research method of the previous researchers: the research method of the gene. Jin and biological materials: the required sample of the sample, the biochemical exploration of 1287581 and the subject matter are reduced, and In the past, the amount of traditional specimens was reduced by more than 2 times. The need for the "dip-dot method", ^ kinetic analysis: with various biological information-related software effects; and not only can achieve full automation The function of the function, the second quantity, the difference between the two basic knives for the search of the gene 匕 更 更 b 父 父 父 父 父 父 父 父 父 父 父 父 于 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物 生物Food inspection, chemical synthesis Drug discovery, early contention on Lu ... and other areas. Class: However, until now, the development of its gene chip has been divided into two major production days (four) wafers: the general glass wafer is fixed on the wafer by a special square, and arranged neatly into p 'Clothing is the required genetic chip; this kind of wafer can not be used in related fields. The main reasons are as follows: , c high, the threshold of large glass processing technology is high, and the cost is relatively high. 匕 It is not a general laboratory or The testing unit can afford it. (2) Fluorescence coloration and hybridization reaction used in glass carrier wafers. • High quality requirements of sample (ribonucleic acid) and light reaction steps. 1 The faulty cursor (cy3 and cy5) is costly and difficult to use. Fix j 3) The result of the fluorescence coloration analysis, the scanner required is a special scanner, and the cost is not high enough for the general experimental unit.龙二)·Nylon film wafer···························· Compared with the glass wafer; the nylon mold wafer preparation 1287581 Niu Yi μ household, the low technical threshold, and the chemical coloring method used: the simple method, the low cost of the tanning agent, but the sample required for this method (Nuclear, Tang nuclear is large, chemical color analysis results without automated analysis, = leading to serious impact on experimental recording and accuracy; therefore, the nylon mold wafer is gradually replaced by glass-ion wafers. ^ In addition to this Whether the glass wafer or the nylon mold wafer is prepared, the dot matrix device is used, and the price of the automated dot matrix device is also quite 叩, and the operation of the instrument is not easy. In the current situation, even if the wafer inspection can be operated: The research laboratory is also unable to independently prepare the required wafers, and must be transferred to a specific biotechnology company for execution; therefore, the above-mentioned conventional gene chip preparation method does not meet the needs of actual use. 1287581 SUMMARY OF THE INVENTION Therefore, the main object of the present invention is to enable a biochip wafer technology platform that is low in cost, easy to operate, and highly efficient by the method of the present invention. The function and application of the present invention can be effectively utilized. Another object of the present invention is to achieve the practical application of the gene wafer to various related fields by the method of the present invention. For the above purpose, the present invention is a microcellular nucleic acid. A method for detecting a wafer, comprising the steps of: a. selecting a nylon mold wafer, using a manual dot-trigger to arrange the desired nucleotide fragments in a neat array on the wafer; b. drying naturally after manual dot-dot, Then, using a nucleic acid fast fixer (cross_linker), the nucleotide fragment is fixed on the nylon mold to complete the wafer preparation; c. collecting the general blood as the sample; d. extracting the ribonucleic acid in the blood and linearly Amplification; e_reverse transcription assay synthesizes a sufficient amount of complementary deoxyribonucleic acid (cDNA) and marks it to form a probe as a probe; f. probes the above-mentioned wafers and markers Hybridization and post-hybridization treatment; g. chemically coloring the wafer after the hybridization reaction with the marker H_ Automated analysis of the image results of the above chemical color reaction. 1287581 ί If, the above-mentioned steps are highly efficient, the gene wafer operation technology is flat. The cost is low, the operation is easy, and the application can be effectively utilized. It is true that W's work is in various related fields. 曰: The practical application of the earth-based wafer [Implementation j, please refer to the "1st, 2nd, 2nd, 2nd, and 闰" diagrams, which is the flow of the present invention. The volume of the cube is not thought of, the manual dot-up + + 铖 铖 铖 铖 铖 铖 铖 铖 铖 铖 铖 铖 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体 立体The method for detecting a wafer by a nucleic acid, the steps comprising: selecting a nylon mold wafer, and arranging each of the gene cores with a size of (10) n丨 and a 15_ pitch lattice on the wafer by using a manual dot machine i, and The desired gene core (four) fragment is made into a secondary blisters of 200 μΜ and arranged in a matrix of 100 n丨 on the wafer, and the manual dot machine 1 (as shown in FIGS. 2 and 3) includes a base.丄, mouth is placed on the base 1 1 The wafer placement layer 1 2, a wafer fixing layer 13 disposed on the wafer placement layer 12, and a dot layer 14 disposed on the wafer fixing layer 13, the wafer placement layer 及2 and the dot layer One side of the 1 4 is connected by a mezzanine hub 15 to enable the opening layer 14 to be opened, and the appropriate layer of the layer 14 is provided with a starting handle 141. A plurality of holes 142 are disposed in the center of the striking layer 14 , and a block of 4·5×4·5 cm is selected at the center of the striking layer 14 and is prepared at a pitch of 3 mm. One row (row: 14; column: 14) hole with an inner diameter of 1 2 mm 1 1287581 4 2 'After the micro-suction tube is inserted (ie, at the dot); the manual tapping machine is used as follows: The starter handle 141 of the active switch 1 is opened to occupy the layer 14 and the wafer is placed on the wafer placement layer i 2 . —2· Cover the dot layer 14 , the wafer fixing layer 13 automatically fixes the wafer to the oligo-nucleic acid, and directly penetrates the hole in the central layer of the layer 1 4 2 Oligonucleotides can be used. b. After the manual dot-drying, it is naturally dried, and then the nucleic acid fast fixator day (cr〇ss-lmke"), the core: acid fragment is fixed on the nylon mold, that is, the preparation of the nucleic acid fast fixer is completed. The high-energy energy of 12QQ Joule is used to join the parent fork, and the nucleotide fragment is fixed on the nylon mold; C. The general blood is collected as the sample, and only 1 c c of blood is collected when collecting the sample. The sample can be reacted to obtain 40 units of cDNA (complementary deoxyribonucleic acid), and only 4 units of cDNA are required for each wafer experiment; d_ extracting the ribonucleic acid in the blood and linearly amplifying; e· The reverse transcription assay synthesizes a sufficient amount of cdNA and marks it to form a probe as a probe; f. performs probe-marking reaction, hybridization reaction, and post-hybridization reaction on the above-mentioned wafer and the target substance. (p〇s 卜 Hybr1dizat•丨〇n) treatment, the probe labeling reaction time and hybridization reaction time is 24 hours; g· chemical reaction of the wafer after the hybridization reaction with the marker is 1287581, after the hybridization When the reaction is processed, its detection The detection time is 30 minutes, which can accelerate the color reaction of the reaction after the hybridization, so that the background color is not excessively colored due to excessive color formation, and the reaction time can be reduced. False Positive Reaction), and after the hybridization and the membrane cleaning before the fixing step, a self-adapting Washing buffer can be used, and the pre-coloring film (Membrane) can be self-adjusted. Detection buffer; h. Automated analysis of the image results after the above chemical color reaction, and the image results after the color reaction can be automatically analyzed by the general density (Density) analysis software. First, we first analyze the experimental results with the Statistical Package for the Social Sciences Ver. 10.0 and learn from the Receiver Operating Characteristics curve that it is determined on each wafer used for this time. After comparing 22 genes with normal gene expression values, if there are 11 or more genes with more than twice the excess table , that is, positive reaction (positive, ten), when 11 or less, it is determined that the wafer is negative (negative, one), so that the most reasonable statistics will be obtained, and the sensitivity and specificity will fall at the most appropriate point. Therefore, this data is used as the standard for interpretation. Please refer to Figure 5, in order to confirm the feasibility and sensitivity of this method, we specially carry out the experiment in the glass, and the K-ras carcinogenesis established before the first 1287581 After the gene-stable transfected adrenocortical cell (K-ras MSTAC) is placed in normal blood, the wafer is analyzed by reacting with the detection wafer of the present invention. Positive control (+) was used and the positive control system employed β-actin 16. As a result, when there are 22 genes on the wafer and 20 K-ras MSTAC cells are contained in the blood of 1c.c., 16 genes are overexpressed, and the wafer is interpreted as a positive reaction, that is, 72.72 The % gene showed positive (+), greater than 50%; and when 1c_c contained 5 K-ras MSTAC fine monthly packets, 'Bei U had 12 genes overexpressed'. This wafer was interpreted as a positive reaction, that is, there was 54.55% of the genes showed positive and greater than 50%; but when the number of cells was reduced to 2, only 3 genes were overexpressed, the wafer was interpreted as a negative reaction, that is, only 13.64% of the performance was positive and less than 50%; when the number of cells is only one, the number of over-expressed genes is less than three and less than 50%, so the result is still interpreted as a negative reaction. Therefore, the detection of the wafer of the present invention is indeed feasible and as long as the number of blood cells per c.c is greater than 5, our detection wafer can accurately detect the activation of the K-ras gene in the blood. Please refer to "Figure 6 and Table 1" for the purpose of detecting the reaction performance of cells in the blood of various cancer patients and specific gene groups by using the micro cell nucleic acid detection wafer technology platform, and the present invention utilizes the instant polymerase chain reaction ( Real-Time PCR) confirms the detection results of the micro-cell nucleic acid detection wafer technology platform. As shown in the figure: wherein the fourth figure is 12 1287581 ^ = : 2 the result of the reaction of the ribonucleic acid of the blood of the cancer patient with the diagnostic day of the present invention; and the eosinophilic because of the instant polymerase chain error "3 is an instant polymerization Enzyme chain-locking reaction ' __ ffl —, ', 贞 reaction is another technique for detecting RNA expression: using the same gene in the diagnostic wafer used in the present invention and whether the gene amplification performance is measured by the K ° _ chain reaction The situation is a magnified filament - it can be seen that the gene detected by the 9 (4) on the wafer is also amplified in the instant polymerase chain reaction.
:…之明可知本發明係具有下列特點： i )手動打點機開發··為解決先前自動化點製機過度 二二,：礙θ及化、操作不易和打點機幫浦不穩定及 軍々以致晶片上基因圓點大小不-之問題，以及, 統圓點墨點法無法同時點製大量核㈣諸於同-尼〕 W…等問題’本發明特別開發操作㈣並能同時打】 大篁基因之手動打點台，可將每—基因核㈣片段』It can be seen that the invention has the following characteristics: i) manual dot machine development ·································································· The problem of the size of the genetic dot on the wafer is not--and the problem of the dot-dot dot method cannot simultaneously produce a large number of cores (4) in the same-ni] W... The special development operation (four) of the present invention can be simultaneously performed. The manual dot-station of the gene can be used for each gene (four) fragment
咖之大小、1.5mm fa1距點陣排列於晶片上，以解》 傳統自動化打點機無法突破之問題。 (2)曰日片製備.為達到晶片廣泛正確應用於各領域，本 發，較尼龍模晶片取代玻璃晶片以解決成本及技術門 仫门等問題，述疋尼龍模晶片後，利用本發明開發之手 動打點機，將所需之基因核芽酸片段以：次水泡製成 2〇〇μΜ以100n丨大整齊排列點陣於晶片上，手動打點後 自然烘乾再以一般實驗用之核酸快速固著器 13 1287581 (cross-linker)，以1200焦耳高能量進行交叉結合 (crosslink)，將核苷酸片段固著於尼龍模上，即完成晶 片製備。 (3) 檢體處理：截至目前為止，因為晶片實驗所需核酸 量且品質要求高大，以目前最普遍被使用的玻璃晶片微 矩陣列分析為例，其所需的核酸之八26〇/八280嚴格要求須 在1·8以上，而所需量須達mRNA2pg (Total RNA 100pg)，以致晶片之實際應用僅侷限於微生物及含大量 核糖核酸的組織檢測，不但檢體取得不易，也阻礙了晶 片的普遍化與發展。 為此本發明改良傳統實驗方式，建立全新之檢體處 理方式，線性放大核糖核酸解決檢體受限之問題並穩定 反轉錄試驗合成足量所需之cDNA，因此即使檢體中之微 量核糖核酸亦可進行晶片反應，目前不僅RNA的品質要 求放寬（僅需 A26〇/A28〇: 1.2)，且僅需 1c_c 的 Whole Blood 即可進行反應得取40單位的cDNA，同時每次晶片實驗 所需之cDNA僅需4單位。 (4) 簡化改良探針標誌反應、雜交反應及雜交後反應試 驗··所有實驗步驟簡易，設備簡單，僅需一部一般定溫 儀器即可完成所有步驟，本發明將探針標誌時間及雜交 時間延長至24小時，更進一步解決微量檢體不易反應的 問題，此外將後處理時偵測（Detection)的時間由傳統 的5〜10分鐘延長至30分鐘，可使之後呈色反應加速， 14 1287581 背景顏色才不至於因過久的成色而造成過度呈色，並且 可降低反應時之誤測（False Positive reaction) 〇 此外本發明所需之固定試劑（Blocking buffer)、清 洗試劑（Washing buffer)及偵測試劑（Detection buff er)等試劑皆可自行泡製使用，而泡製時之調配方式係 以固定試劑（Blocking buffer) : 50g、清洗試劑（Was hing buffer) :0.4% SDS ( Sodium Dodecyl Sulphate， 十二烧基硫酸納）；〇.5%SSC ( Sodium Chloride Sodiu m Citrate，氯化鈉一檸檬酸鈉溶液）、以及偵測試劑（D etection buffer):0_1M (體積莫耳濃度）Tris-HCI (三 氯化氫）；0·1Μ NaCI (氯化鈉）；50mM (體積莫耳濃度 之千分之一）MgCI2.6H2〇（水溶性氯化鎂）所泡製而成； 此外，本發明所使用之偵測（Detection)及二次抗體皆 可回收再次使用，因此一般實驗室及相關單位皆可獨立 完成縮有實驗步驟。 (5)自動化分析：實驗所得之影像結果可利用一般密度 (Density)分析軟體自動分析，而該密度分析軟體可為 Alpha Ease FC Stand Alone V.3.1.2，或 GeneTACT M Integrator Version 3_3等軟體，解決過去呈色型反應 實驗結果判讀不精確之問題，且此相關軟體操作簡易、 成本低；所得之結果亦可依使用者之需求，利用生物資 訊相關軟體進一步研究利用之。 綜上所述，本發明微量細胞核酸檢測晶片之方法， 15 1287581 不僅犬破傳統晶片實驗普及化之困難’將基因晶片實降 f用於各領域中；使得各相關研究或檢測，如··基因： 能及表現分析、基因突變之解析、差異性基因篩選 錄口子之搜哥.··等’基因研究及新藥研發、臨床疾 =驗、㈣追縱、食品檢測、農產品改良暨研發..·、 等貫際應用研發必能更順暢進行。 且可有效改善習用之種種缺點，進而使本發明之産 ϋίί進步i更實用、更符合使用者之所須，確已符合 二主申睛之要件’麦依法提出專利申請，尚請貴 =委貝撥冗細審，並盼早日准予專利以勵發明，實感 德便。 准乂上所34者’僅為本發明之較佳實施例而已，當 限定本：明實施之範圍；故，凡依本發明申請 飾：庫况明書内容所作之簡單的等效變化與修 飾’白應仍屬本發明專利涵蓋之範_。The size of the coffee, 1.5mm fa1 is arranged on the wafer from the dot matrix to solve the problem that the traditional automatic dot-punching machine cannot break through. (2) Preparation of the film. In order to achieve the wide and correct application of the wafer to various fields, the present invention replaces the glass wafer with a nylon mold wafer to solve the problems of cost and technical threshold, and after the nylon mold wafer is described, the invention is developed. The manual dot-punching machine prepares the desired gene nucleotide fragment in a sub-bubble of 2〇〇μΜ and arranges it on the wafer in a large order of 100n丨, and then manually dries and then naturally dries and then uses the nucleic acid for general experiment. Fixer 13 1287581 (cross-linker), cross-linking at 1200 joules of high energy, fixing the nucleotide fragments on the nylon mold, complete wafer preparation. (3) Sample processing: Up to now, because of the high amount of nucleic acid required for wafer experiments and the high quality requirements, the most commonly used glass wafer microarray analysis is used as an example, and the required nucleic acid is 八〇8/8 280 strict requirements must be above 1.8, and the required amount must reach 2pg (Total RNA 100pg), so that the practical application of the wafer is limited to microbes and tissue containing a large amount of ribonucleic acid, not only the sample is not easy to obtain, but also hindered The generalization and development of wafers. To this end, the present invention improves the traditional experimental mode, establishes a new method of sample processing, linearly amplifies the ribonucleic acid to solve the problem of limited specimens, and stabilizes the reverse transcription test to synthesize a sufficient amount of cDNA, so even a small amount of ribonucleic acid in the sample It is also possible to carry out wafer reactions. At present, not only the quality requirements of RNA are relaxed (only A26〇/A28〇: 1.2), but only 1c_c of Whole Blood can be used to carry out 40 units of cDNA, which is required for each wafer experiment. The cDNA requires only 4 units. (4) Simplified and improved probe labeling reaction, hybridization reaction and post-hybridization reaction test··Each experiment procedure is simple, the equipment is simple, all the steps can be completed by only one general temperature measuring instrument, and the invention marks the probe time and hybridization The time is extended to 24 hours, which further solves the problem that the micro-sample is not easy to react. In addition, the detection time during post-processing is extended from the conventional 5 to 10 minutes to 30 minutes, which can accelerate the subsequent color reaction. 1287581 The background color is not excessively colored due to excessive color formation, and the False Positive reaction can be reduced. In addition, the blocking reagent and Washing buffer required by the present invention are further provided. And detection reagents (Detection buff er) and other reagents can be brewed by themselves, and the preparation method is the blocking reagent: 50g, Washing buffer: 0.4% SDS (Sodium Dodecyl) Sulphate, sodium dodecyl sulfate; 5%.5% SSC (Sodium Chloride Sodiu m Citrate, sodium chloride-sodium citrate solution), and detection test (D etection buffer): 0_1M (volume concentration) Tris-HCI (hydrogen chloride); 0·1Μ NaCI (sodium chloride); 50 mM (one thousandth of a molar concentration) MgCI2.6H2 〇 (water soluble The magnesium chloride is brewed; in addition, the detection and secondary antibodies used in the present invention can be recycled and reused, so that the laboratory and related units can independently complete the shrinking experimental steps. (5) Automated analysis: The image results obtained by the experiment can be automatically analyzed by the general density (Density) analysis software, and the density analysis software can be software such as Alpha Ease FC Stand Alone V.3.1.2, or GeneTACT M Integrator Version 3_3. Solve the problem of inaccurate interpretation of the results of past color-induced reaction experiments, and the related software is easy to operate and low in cost; the results obtained can be further researched and utilized by bio-information-related software according to the needs of users. In summary, the method for detecting a micro cell nucleic acid detecting chip of the present invention, 15 1287581 is not only difficult to popularize the traditional wafer experiment of the dog, but the gene chip is used in various fields; so that various related research or detection, such as ··· Genes: analysis of energy and performance, analysis of gene mutations, screening of differential gene screening, search for brothers, etc. 'Gene research and new drug research and development, clinical disease test, (4) memorial, food testing, agricultural product improvement and research and development.. ·, and even the application of research and development will be smoother. Moreover, it can effectively improve various disadvantages of the conventional use, thereby making the invention of the invention more practical and more in line with the needs of the user, and indeed meets the requirements of the second mainstay, and the patent application is filed according to law. Bayer has a rigorous review and hopes to grant patents at an early date to invent inventions. The reference to '34' is only a preferred embodiment of the present invention, and is limited to the scope of the present invention; therefore, the simple equivalent change and modification of the contents of the library: It should still be the scope covered by the patent of the present invention.
16 1287581 【圖式簡單說明】 第1圖，係本發明之流程方塊示意圖。 第2圖，手動打點機之立體外觀示意圖。 第3圖，手動打點機之立體分解示意圖。 第4圖，生物統計軟體分析實驗結果示意圖。 第5圖，K-ras致癌基因穩定轉染之人類腎上腺皮質 細胞株實驗結果示意圖。 第6圖，係本發明利用微量細胞核酸檢測晶片技術平 台檢測各種癌症病人血液中細胞與特定基因 群之反應表現不意圖。 表一：係本發明利用即時聚合酶鏈鎖反應（Rea卜Time PCR)確認微量細胞核酸檢測晶片技術平台檢 測結果。 【主要元件符號說明】 步驟a :手動打點機製備 步驟b:點製基因晶片 步驟c :收集一般血液檢體 步驟d:萃取檢體中之核糖核酸並進行線性放大 步驟e :反轉錄合成c DNA並標誌之 步驟f ··晶片與標誌物雜交（Hybridization)反應以及雜 交後反應（Post-Hybrid ization) 步驟g :化學呈色反應 步驟h:影像結果自動化分析 17 1287581 手動打點機1 底座1 1 晶片放置層1 2 晶片固定層1 3 打點層1 4 啟動手把1 4 1 孔洞1 4 2 夾層樞紐1 5 正控制（+)之β—肌動蛋白1616 1287581 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a block diagram showing the flow of the present invention. Figure 2 is a schematic view of the stereoscopic appearance of a manual dot-punching machine. Figure 3 is a perspective exploded view of a manual dot machine. Figure 4 is a schematic diagram of the results of biostatistical software analysis experiments. Fig. 5 is a schematic diagram showing the results of human adrenocortical cell lines stably transfected with K-ras oncogene. Fig. 6 is a view showing the use of the microcellular nucleic acid detection wafer technology platform to detect the reaction performance of cells in the blood of various cancer patients and specific gene groups. Table 1: The present invention uses the real-time polymerase chain reaction (Rea Time PCR) to confirm the detection results of the micro-cell nucleic acid detection wafer technology platform. [Description of main component symbols] Step a: Manual dot-making machine preparation step b: Point-made gene wafer Step c: Collecting a general blood sample Step d: Extracting the ribonucleic acid in the sample and performing linear amplification step e: Reverse transcription synthesis of c-DNA And mark step f ·· wafer and marker hybridization (Hybridization) reaction and post-hybridization reaction (Post-Hybrid ization) Step g: chemical color reaction step h: image analysis automated analysis 17 1287581 manual dot machine 1 base 1 1 wafer Placement layer 1 2 wafer fixed layer 1 3 Dot layer 1 4 Start handle 1 4 1 Hole 1 4 2 Sandwich hub 1 5 Positive control (+) β-actin 16
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