TW200806799A

TW200806799A - Rapidly analysis method for nucleic acid blot

Info

Publication number: TW200806799A
Application number: TW95126029A
Authority: TW
Inventors: Chung-Cheng Chang; Jau-Der Chen; Bo-Tai Chen
Original assignee: Chung-Cheng Chang; Jau-Der Chen; Bo-Tai Chen
Priority date: 2006-07-17
Filing date: 2006-07-17
Publication date: 2008-02-01
Also published as: TWI326711B

Abstract

This invention discloses an rapid analysis method for nucleic acid blot. Firstly the nucleic acid to be analyzed is impressed on a base substance and the nucleic acid prepared for analysis is immobilized on the base substrate. After that, the blocking process of the prehybridization which is commonly needed is omitted. Under the situation that the area in which the nucleic acid prepared for analysis is not immobilized on the base substrate is not under the blocking condition, the nucleic acid probe is directly added to carry out the hybridization for a short period and finally to carry out the cleaning process to separate and eliminate the nucleic acid probe which is not combined with the nucleic acid prepared for analysis. And the testing result is obtained through the follow-up testing process of hybrid signals. By means of eliminating the prehybridization process and shortening the time of the hybridization and the cleaning reaction of this invention, the required reaction time of nucleic acid hybridization is greatly shortened that may rapidly complete the analysis process of nucleic acid blot.

Description

200806799 九、發明說明：【發明所屬之技術領域】本發明係關於一種核酸分析方法，尤其是關於一種直接將待分析核酸傳遞至一基質上，並能夠縮短核酸雜交 (hybridization)所需反應時間之快速核酸印潰（blot)分析方法。【先前技術】 / 在一般基因選殖（cloning)之操作過程中，為確認基因或DNA片段是否正確選殖至載體中，除了以膠體電泳 (gel electrophoresis)來分析DNA之分子量大小外，更精確的確認方式即是利用一探針（pr〇be )核酸與待確認之200806799 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a nucleic acid analysis method, and more particularly to a method for directly transferring a nucleic acid to be analyzed to a substrate and shortening the reaction time required for nucleic acid hybridization. Rapid nucleic acid blot analysis method. [Prior Art] / In the process of general gene cloning, in order to confirm whether the gene or DNA fragment is correctly selected into the vector, in addition to analyzing the molecular weight of DNA by gel electrophoresis, it is more accurate. The method of confirmation is to use a probe (pr〇be) nucleic acid and to be confirmed

DNA進行雜交反應（hybridization)，藉以分析待測DNA 是否選殖有所欲之基因或DNA片段。同樣的情況，若係在研九基因的表現，則轉錄（tranSCripti〇n )後所產生之 mRNA ’其長度大小以及表現量的多寡，則是基因表現後必須偵測之重要指標，而其枝亦___探針滅The DNA is subjected to hybridization to analyze whether the DNA to be tested is selected for a desired gene or DNA fragment. In the same situation, if the expression of the Nine Gene is transcribed, the length of mRNA and the amount of mRNA produced after transcription (tranSCripti〇n) is an important indicator that must be detected after gene expression, and its branch Also ___ probe out

再藉由雜交訊號結果分析RNA 與該RNA進行雜交反應，之長度或複本（copy)數。印潰分析方法一一般係對薄膜上所吸附之待分析核The hybridization signal results are used to analyze the length of the hybridization reaction of RNA with the RNA, or the number of copies. The method of the analysis of the crushing method is generally the adsorption of the core to be analyzed on the film.

酸，以具專一性（specii 進—步藉*控AL 一 200806799 滴放於薄社’使紐料韻其上。較若泳膠體轉印至_上之DNA與探針進行雜妖應者，稱之為南方轉潰法（SGUthemblQtting)，若係對轉印之rna 進行雜交反應者，則稱之為北方轉潰法（N〇rthem blotting) ’其他錢棘赠分析贿者，㈣滴放之範圍而分別有點狀、細紋狀與塊狀之印潰法（齡/ sl〇t^sp〇t blotting )其中，點狀、細紋狀與塊狀等印潰法，因待分析核酸無_過電狀分離，也錢進行轉印之步驟，故可縮短操作分析所需之時間，且因其同時無須利用到電泳、轉印設備與相關的溶液試劑，在實驗成本上亦較為經濟，故常用在一般定性分析或大批次的檢測分析上。請參閱第一圖，該圖係習知點狀印潰法之流程示意圖。整個印潰分析方法，約略可分作薄膜前處理si〇、待分析核酸處理S20、核酸雜交反應S3〇與雜交訊息的偵測 S40。首先將薄膜處理後，將待分析核酸滴放其上，而後經過核酸雜交配對反應，最後對雜交訊號進行檢測，即完成整個點狀印潰分析流程。薄膜前處理步驟S10，其係先提供一可吸附待分析核酸之薄膜S11，而後再將該薄膜加以浸潤S12，使該些薄膜完全濕潤，而能進一步滲透待分析核酸並使其吸附。目前常見的薄膜有尼龍膜（nylon membrane)與硝化纖維紙 (nitrocellulose membrane)，而其浸潤方式則先將薄膜完王/叉濕於去離子水（deionized water)後，再浸潤於6至 10倍之標準檸檬酸納溶液[stancjard s〇cjium cerate，簡稱 200806799 SSC ; 20 倍 SSC 包括 3 Μ 氣化鈉（sodillm cW〇ride )與〇·3 Μ ’ pH 7·0 檸檬酸納（sodium citrate)]中備用。進行雜父反應说，無論是DNA或是RNA待測樣本，皆須經過變性（denaturation)的步驟，使其保持在單股 (single chain )狀態，以能與單股的探針相配對 (annealing)。因此，在待分析核酸處理S2〇步驟中，於製備待分析核酸S21步驟後，需先經步驟S22，將該待分析核酸加以k性，然後於步驟S23中滴放待分析核酸樣央於該薄膜上。待分析核酸吸附於薄膜上後，進一步須將該核酸固定於薄膜上，使該核酸於後續雜交反應時不會脫落。習知核酸固定的方式，可經步驟^々，於肋艽下烘乾 30分鐘至2小時，或是經步驟S25，利用波長254 nm之紫外光照射，使核酸分子與該薄膜之纖維間形成共價交聯 (crosslink)，而將核酸分子固定其上。將待分析核酸固定於薄膜上後，接著進行核酸雜交反應步驟S30。由於薄膜上未固定有待分析核酸之區域，仍可吸附核酸，若不將該些區域加以阻斷（bl〇cking)，則加入同為核酸之探針時，探針將非專一性地結合於薄膜之該些區域上，因此進行雜交反應前需先經一前雜交反應 (prehybddization)步驟S3卜而於薄膜上加入含有5倍 SSC 溶液、50%(v/v)甲醯胺（formamide)、〇·〗％(_)硫酸十二酯鈉溶液（sodium dodecyl sulfate，簡稱 SDS)、5 %酪蛋白（casein)之溶液，於42°C下反應1至2小時，將未固定有待分析核酸之區域加以阻斷蓋滿。 200806799 之後’即可進行步驟S32之雜交反應，將標記有酵素或放射線元素等之探針核酸加至前述經前雜交反應之薄膜上，而後於42°C下反應16小時以上。此期間探針會尋找互補的待分析核酸加以配對。其後，進行清洗反應&驟 S33 ’將未配對結合的探針核酸沖洗掉。清洗時，可利用含有2倍SSC溶液與〇.l%(w/v) SDS的清洗溶液於^溫下漂洗5分鐘，共漂洗二次。然後再以68艺，含有〇1倍ssc 與0.1%(w/v) SDS的清洗溶液漂洗二次，每次ι5分鐘。經過清洗反應S33後，薄膜上只剩能夠與待分析核酸相配對的探針，此時即可進行雜交反應訊號之檢測步驟 S40。進行雜交反應訊號檢測時，可配合探針所標記之顯示分子，而使用適當的檢測方式。常見的檢測方式有利用 DIG(dig〇xigenin)系統之呈色反應檢測方式S41，標記以放射線元素32P或35S之放射線顯影檢測方式S42，以及利用HEX、Cy3及Cyr之冷光反應檢測方式S43。由前述步驟觀之，習知印潰分析方法，無論係薄膜前處理S10、待分析核酸處理S20或是核酸雜交反應S30等步驟’皆需經歷相當多之操作流程，而其所需耗費之操作曰可間亦相當長’若加上雜交訊號偵測S4〇的步驟，大約需要二天才能完成。因此，對於許多急於知曉檢測結果的試 ^即热法於短時間内完成。此外’對於一些較簡單的核酉文疋性檢測，若仍須花費相當多時間與試劑去試驗，亦是相當不經濟。因此，研究一快速，能夠簡化印潰分析所需步驟與時間，並同時兼顧低背景雜訊之印潰分析方法，對 200806799 於一般簡易檢測或是大批次檢測，都可有效縮短試驗所需時間，並同時大幅降低所需耗費之材料成本。【發明内容】為簡化印潰分析方法所需之操作步驟，同時減少操作試驗時所需之·、溶液，並縮短整個分制需耗費之時間’本發明提供-種將習知祕交反應等步驟加以省略，並能大幅縮短滅印潰分析所f時間之快速核酸印潰分析方法。本發明之快速減印潰分析方法，省略習知前雜交反應步驟所必經之阻斷作用，並利基於相當短時間内即可完成配對之雜，將探針_與待分析核酸進行配對反應以及清洗反應所需之_相對應調整驗，進而使整個印潰分析紐職之_大幅触，使能於數十分鐘内，完成待分析核酸處理至核酸雜交反應之步驟流程，而達到節省試驗時間，並同時減少相關試劑、溶液等成本支出之目的。曰本發明之快速核酸印潰分析方法，包括下列步驟：⑴ ，供基質，該基質係具有孔洞；⑺將—待分析核酸傳遞至該基質上，並使該待分析核料該基f所㈣；⑺將，待分析核_定於該基質上；（4)於不將該基質上未固定有該待分析核酸區域加以阻斷（blGddng)之情兄下，將-含有探針（_〇概之溶液加人步驟(3)的該基質上’使該騎減與絲質上之該齡析_騎驗基配對（base pairing)數分鐘；（5)將步驟(4)中未與該待分析 200806799 核酸相結合的該探針核酸分離去除；以及(6)檢測經步驟 (5)之該基質上的雜交訊息。其中’步驟(1)中之基質可為薄膜（membrane)、矽晶片、玻璃、磁珠或金屬粒子，但並不僅限於此。因此當將待分析核酸傳遞至該基質後，於步驟(2)與步驟(3)中，/待分析核酸可藉由乾燥吸附、電性吸附、磁性吸附等方式吸附於該基質上’其吸附方式亦不僅限於前述。而於後續之步驟(4)中，待分析核酸則可藉由乾燥、加熱烘乾、紫外光照射或磁吸等方式固定於該基質上，所述固定方式同樣僅係例示。藉由鹼基配對時氫鍵迅速產生配對連結之特性，無法配對之探針核酸即可被分離去除，因而可省略習知雜交處理過程所需的部分步驟，並縮短習知漫長反應所需之等待時間。本發明同時提供另一種快速核酸印潰分析方法，包括下列步驟.（1)提供—薄膜’該薄膜可呈乾燥狀；⑺將 :待分析核酸傳輕該薄耻，並使該待分析核酸為該薄膜所吸附，（3)將該待分析核g㈣定於該薄膜上，之後可乾燥該薄膜；（4)於不將該基質上未固定有該待分析核酸區域加以_之情況τ ’將—含有探針核酸之溶液與經步驟⑶=_於贼至贼下相互作用，使該探針核酸與該薄膜上之該待分析猶進行驗基配對2至將經步驟⑷之該薄膜，以緩衝溶液漂洗，私將未盘該\寺分析核酸相結合㈣騎㈣絲去除3 步驟(5)之該薄膜上的雜交訊息。冬 200806799 其中，本發明所得檢測之待分析核酸樣本可為dna 或RNA，而該步驟⑴中之該薄膜則可為尼龍膜（nylon membrane)或硝化纖維紙（nitr〇ce丨論％咖_咖），但並不以此為限。尼龍膜可帶有正電荷或不帶電，尼龍膜盥确化纖維紙之孔洞餘可為G.1至50 ,之間，其餘待分析核酸分子量大小而選擇適用之孔徑大小，核酸蒼大時所使狀孔徑也愈大，其中以〇·2 μιη與〇·45 _為較佳。此外’薄膜可不經制之前處理步驟，而係呈乾燥狀，使將來滴放待分析鋪時可迅速使其韻於該㈣上。步驟〇)中，可將該吸附有待分析核酸之薄膜，置於8〇。〇至13〇下火、乾、力1至1〇分鐘，藉以將待分析核酸固定於薄膜上其中，烘乾溫度以100至120°c，為較佳。此外亦可利用各外光（UV)照射方式將待分析核酸固定於薄膜上。珂述利用紫外光固定待分析核酸後，尚可進一步利用烘乾等方式將薄膜乾燥化。步驟(5)中該緩衝溶液以低離子強度者為較么，其可包括〇·〇5至〇」5倍之標準檸檬酸納溶液，與0.05%至〇.i5%(w/v)之硫酸十二酯鈉溶液，其中以〇1 七之標準檸檬酸鈉溶液，與0.1%(w/v)之硫酸十二酯鈉溶液為較佳。漂洗時間則可在3至6分鐘之間。藉由本發明之快速核酸印潰分析方法，薄膜之前處理乂 ~係有別於習知先將薄膜浸潤後再加以烘乾固定之步驟’而係直接利用乾燥之薄膜滴放待分析核酸使其吸收。由於核酸於薄膜乾燥狀況下比於濕潤狀況下擴散更為迅速’故該待分析核酸即可迅速為薄膜所吸附，因而可縮短 11 200806799 處理所需之時間，以及相關步驟所需之設備、溶液、於4膜上滴放朗定崎分析核酸後，可利用方式使j薄膜乾燥化，其後即可進行雜交反應。進行雜六反應之前，習知分析方法需㈣薄膜上未固定有待分^ 酸之區域以含有阻斷溶劑（blocking reagent)之溶液所覆盖，糟以避免將來探針非專一性的結合。然而，於本發明方法中，並不需經過前述之前雜交反X應 (^rehybruhzation )，而係於薄膜上直接加入探針核酸進行雜交反應，並僅反應數分鐘即可完成。其·在於，當將，酸溶液加入至呈乾燥狀之薄膜後，該探針核酸：液 =迅速為薄膜微細孔洞所產生之毛細現象所吸引，相較於白知之濕潤狀態，將更快速為薄膜所吸收，並進入薄膜内 j之孔洞中。探針核酸溶液中之核酸係以布朗運動方式於薄膜内移動並同時尋找可轉之待分析賊，以長度為3〇個核普酸（3〇_mer )之DNA於？ μπι薄膜孔洞中移動為例，其擴散時間僅需〇·6秒，而5000-mer之DNA亦僅需25秒。同守依據小所周知之聚合酶連鎖反應（p〇lymerasechah PCR)’引子與樣本核酸之配對（annealing)時間僅需不到1分鐘即可完成。因此，於本發明之條件下，探針核酸能在相當短的時間内完成驗基配對，因而能夠大中田縮短習知雜交反應所需花費十數小時之時間，而於數分鐘内完成。另一方面，如前所述，探針核酸對待分析核酸之配對 12 200806799 二藉#下7 (pUrine)或口密咬（pyrimi出狀）氫鍵之結合力，、而此氫鍵之形成係相當迅速，亦比探針核酸吸附至薄膜之速度陕^日守挺針核酸與待分析核酸之配對結合力係比探針核酸與薄膜間之吸附力強，而待分析核酸與薄膜間之吸附力亦”木針核酸對薄膜之吸附力為大，域針核酸係較一不似大分子的待分析核酸係卡陷在薄旗之孔洞中’因此於後續之清洗反應中，可輕易清洗掉與薄膜非專-性結合之探針核酸，而呈現出低背景雜訊之雜交反應、…果.而此亦係$庸進行前雜交反應阻斷步驟之原因。但為避免原本刻定之待分析核酸於清洗_料，清洗反應Mxk者細鋪子@紅溶液洗的時間餘分鐘即可，其帽佳者為4至6分鐘。’下，漂洗後之雜交反應薄膜則可進行下—步驟關於雜父反應喊之_。雜交反應訊號_可_目前已知之 ^方Γΐ套（kit)進行，並可快速之光電檢測方式細糾斷間至數分鐘内。所述光電檢财式可包括：阻抗檢測、電容值檢測、電阻值制或電化學檢不以此為限。、 /宗上所述’藉由本料之快速_印潰分析方法，益論係步驟⑴至步驟⑶之待分析核酸處理或是步：驟(5)之核酸雜交反麟步驟，皆僅需花紐分鐘 1 加總上述步驟，也總共僅需花㈣15錢分鐘，相較於傳統方法，係大幅並有效改善核酸印潰分析方法所需之時間與材料成本。 ' 200806799 以下將配合圖式進一步說明本發明的實施方式，下述所列舉的實施例係用以闡明本發明，並非用以限定本發明之範圍，任何熟習此技藝者，在不脫離本發明之精神和範圍内，當可做些許更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。【實施方式】請參閱第二圖，該圖係本發明實施例之流程示意圓^ ^發明快速核酸印潰分析方法之實施例中，包括待分析核酸處理S50、核酸雜交反應S6〇與雜交訊息的偵測s4〇等步驟。盲先將待分析核酸傳遞至基質上，並進行待分析核 ^固定，錢不經祕交步驟而直接.騎待分析核酸之 τ隹父配對反應’最後對雜交訊號進行彳貞測，即可^成本發明核酸印潰分析之步驟流程。明、、fe、、、良芩閱第二圖，待分析核酸處理步驟“Ο中，包括於S51中提供一具孔洞之基質；於步驟中將為，52G中所製備之待分析核酸傳遞至該基質上，同時使顧刀析核酸為該基質所吸附；於步驟s53中則將該待 =核酸固定於該基質上。其中，步驟S51中之基質可為 ’專膜membrane)、石夕晶片、玻璃、磁珠或金屬粒子，但並不僅限於此。目此當將待分析核酸傳it至該基質後，於 f驟S52與步驟S53中，待分析核酸可藉由乾燥吸附、電 '生吸附、磁性’等方式韻於絲質上，其Acid, with specificity (specii, step by step, control, AL, 200806799, drop in the thin society, so that the material is on the rhyme. If the swimming gel is transferred to the DNA and the probe is used for the demon, It is called the Southern Stroke Method (SGUthemblQtting). If it is a hybrid reaction to the transferred rna, it is called N〇rthem blotting. 'Other money is used to analyze the bribe. (4) Drip. Scope and a little bit, fine striate and block-like printing method (age / sl〇t^sp〇t blotting), point, fine striate and block-like printing method, because the nucleic acid to be analyzed is not _ The electrical separation and the transfer step are also carried out, so that the time required for the operation analysis can be shortened, and since it is not necessary to utilize the electrophoresis, the transfer device and the related solution reagent, the experimental cost is also economical, so It is commonly used in general qualitative analysis or large batch detection analysis. Please refer to the first figure, which is a schematic diagram of the flow of the conventional point-like printing method. The whole printing and analyzing method can be roughly divided into film pretreatment si〇, waiting Analysis of nucleic acid treatment S20, nucleic acid hybridization reaction S3〇 and hybridization information S40 is measured. After the film is processed, the nucleic acid to be analyzed is dropped thereon, and then subjected to nucleic acid hybridization pairing reaction, and finally the hybridization signal is detected, that is, the entire dot-shaped crushing analysis process is completed. The film pre-processing step S10 is performed. First, a film S11 capable of adsorbing the nucleic acid to be analyzed is provided, and then the film is infiltrated with S12 to completely wet the film, and the nucleic acid to be analyzed is further infiltrated and adsorbed. The common film currently has a nylon membrane (nylon membrane). ) and nitrocellulose membrane, and the method of infiltration is to wet the membrane after deionized water, and then infiltrate 6 to 10 times the standard sodium citrate solution [stancjard s〇 Cjium cerate, referred to as 200806799 SSC; 20 times SSC includes 3 Μ gasified sodium (sodillm cW〇ride) and 〇·3 Μ 'pH 7·0 sodium citrate】 in standby. It is a sample of DNA or RNA to be tested, which must be subjected to a denaturation step to maintain it in a single chain to be paired with a single-stranded probe ( Therefore, in the step of preparing the nucleic acid to be analyzed, in the step of preparing the nucleic acid S21 to be analyzed, the nucleic acid to be analyzed is subjected to k-sexation through step S22, and then the nucleic acid to be analyzed is dripped in step S23. After the nucleic acid to be analyzed is adsorbed on the film, the nucleic acid is further immobilized on the film so that the nucleic acid does not fall off in the subsequent hybridization reaction. The nucleic acid immobilization method can be carried out by drying the ribs for 30 minutes to 2 hours, or by step S25, using ultraviolet light having a wavelength of 254 nm to form a nucleic acid molecule and the fibers of the film. Covalently crosslinks, and nucleic acid molecules are immobilized thereon. After the nucleic acid to be analyzed is immobilized on the film, the nucleic acid hybridization reaction step S30 is followed. Since the nucleic acid is not immobilized on the membrane, the nucleic acid can be adsorbed. If the region is not blocked, the probe will be non-specifically bound when the probe is the same as the nucleic acid. In the regions of the film, it is necessary to add a 5-fold SSC solution, 50% (v/v) formamide, to the film by a prehybddization step S3 before performing the hybridization reaction. 〇·〗〖% (_) sodium dodecyl sulfate (SDS), 5% casein (casein) solution, reacted at 42 ° C for 1 to 2 hours, will not be fixed with the nucleic acid to be analyzed The area is blocked and covered. After the hybridization reaction of the step S32, the probe nucleic acid labeled with an enzyme or a radiation element or the like is added to the film of the pre-hybridization reaction, and then reacted at 42 ° C for 16 hours or more. During this time, the probe will look for complementary nucleic acids to be analyzed for pairing. Thereafter, a washing reaction &S33' is performed to rinse off the unpaired bound probe nucleic acid. For cleaning, it can be rinsed for 5 minutes at a temperature of 2 times with a cleaning solution containing 2 times SSC solution and 〇.l% (w/v) SDS, and rinsed twice. Then rinse again twice with a cleaning solution containing 〇1 times ssc and 0.1% (w/v) SDS for 5 minutes each time. After the cleaning reaction S33, only the probe capable of pairing with the nucleic acid to be analyzed remains on the film, and at this time, the detection step S40 of the hybridization reaction signal can be performed. When performing hybridization reaction signal detection, an appropriate detection method can be used in conjunction with the display molecule labeled by the probe. Common detection methods include the color reaction detection method S41 using the DIG (dig〇xigenin) system, the radiation development detection method S42 marked with the radiation element 32P or 35S, and the cold light reaction detection method S43 using HEX, Cy3 and Cyr. According to the foregoing steps, the conventional printing and analyzing method, whether it is the film pretreatment S10, the nucleic acid treatment S20 or the nucleic acid hybridization reaction S30, etc., has to undergo a considerable number of operation processes, and the required operation thereof It is also quite long. If you add the hybrid signal to detect S4, it takes about two days to complete. Therefore, for many tests that are eager to know the test results, the thermal method is completed in a short time. In addition, it is quite uneconomical for some simpler nuclear testes to take considerable time and reagents to test. Therefore, the research is fast, can simplify the steps and time required for the analysis of the ink, and at the same time take into account the low-background noise detection method, which can effectively shorten the test time for the 200806799 general simple test or large batch test. And at the same time drastically reduce the cost of materials required. SUMMARY OF THE INVENTION In order to simplify the operation steps required for the crushing analysis method, at the same time, it is necessary to reduce the time required for the operation test, and to shorten the time required for the entire dispensing system. The present invention provides a conventional secret reaction, etc. The steps are omitted, and the rapid nucleic acid crushing analysis method of the time of the extinction analysis can be greatly shortened. The rapid subtraction analysis method of the present invention omits the blocking effect of the conventional pre-hybridization reaction step, and the pairing is completed based on a relatively short time, and the probe_ is paired with the nucleic acid to be analyzed. And the corresponding adjustment test required for the cleaning reaction, so that the entire printing and analysis of the new position of the _ large touch, enabling the process of the nucleic acid to be analyzed to the nucleic acid hybridization reaction in a few tens of minutes, to achieve the savings test Time, and at the same time reduce the cost of related reagents, solutions and other costs. The method for rapid nucleic acid fragmentation analysis of the present invention comprises the steps of: (1) providing a matrix having pores; (7) transferring the nucleic acid to be analyzed to the substrate, and allowing the substrate to be analyzed to have the base f(4) (7) The core to be analyzed is set on the substrate; (4) the probe containing the nucleic acid to be analyzed is not immobilized on the substrate (blGddng), and the probe is contained (_〇) The solution is added to the substrate of step (3) to 'make the ride minus the age of the silky pairing base pairing for several minutes; (5) the step (4) is not The probe nucleic acid combined with the 200806799 nucleic acid is separated and removed; and (6) detecting the hybridization message on the substrate according to the step (5), wherein the substrate in the step (1) can be a membrane or a germanium wafer. , glass, magnetic beads or metal particles, but not limited to this. Therefore, after the nucleic acid to be analyzed is transferred to the substrate, in steps (2) and (3), the nucleic acid to be analyzed can be dried by adsorption, electricity Adsorption, magnetic adsorption, etc. are adsorbed on the substrate. In the subsequent step (4), the nucleic acid to be analyzed may be immobilized on the substrate by drying, heating and drying, ultraviolet light irradiation or magnetic attraction, etc., and the fixing manner is also merely exemplified. Hydrogen bonds rapidly create a paired link during base pairing, and probe nucleic acids that cannot be paired can be separated and removed, thereby eliminating some of the steps required for conventional hybridization processes and shortening the waiting time required for conventional long reactions. The invention also provides another rapid nucleic acid crushing analysis method, comprising the following steps: (1) providing - the film 'the film can be dry; (7): the nucleic acid to be analyzed is lightly shameful, and the nucleic acid to be analyzed For the adsorption of the film, (3) the core g(4) to be analyzed is fixed on the film, and then the film can be dried; (4) the case where the nucleic acid to be analyzed is not immobilized on the substrate τ' Combining the solution containing the probe nucleic acid with the step (3) = _ thief to the thief, so that the probe nucleic acid is paired with the analyte to be analyzed on the film 2 to the film which will be subjected to the step (4), Buffer The solution is rinsed, and the hybridization message on the film of the third step (5) is removed by the combination of the four-step analysis nucleic acid (4) riding (four) silk. Winter 200806799 wherein the nucleic acid sample to be analyzed obtained by the present invention may be dna or RNA. The film in the step (1) may be a nylon membrane or a nitrocellulose paper (nitr〇ce丨% coffee_coffee), but not limited thereto. The nylon membrane may have a positive charge or Without charge, the pores of the nylon membrane can be determined to be between G.1 and 50, and the size of the remaining molecular weight of the nucleic acid to be analyzed is selected, and the pore size is larger when the nucleic acid is large. It is preferable to use 〇·2 μιη and 〇·45 _. In addition, the film can be dried without being subjected to a pre-treatment step, so that it can be quickly scented on the (4) when it is to be deposited in the future. In the step 〇), the film to which the nucleic acid to be analyzed is adsorbed can be placed at 8 Torr. 〇 to 13 〇 Under fire, dry, and force for 1 to 1 minute, whereby the nucleic acid to be analyzed is fixed on the film, and the drying temperature is preferably 100 to 120 ° C. Alternatively, the nucleic acid to be analyzed may be immobilized on the film by means of external light (UV) irradiation. After the nucleic acid to be analyzed is fixed by ultraviolet light, the film may be further dried by drying or the like. In the step (5), the buffer solution is preferably a low ionic strength, which may include a standard citrate solution of 5 times 〇·〇5 to 〇, and 0.05% to 〇.i5% (w/v). A sodium dodecyl sulfate solution in which a standard sodium citrate solution of 〇1-7 is used, and a 0.1% (w/v) sodium dodecyl sulfate solution is preferred. The rinse time can be between 3 and 6 minutes. According to the rapid nucleic acid fragmentation analysis method of the present invention, the film pretreatment is different from the conventional step of infiltrating the film and then drying and fixing, and the nucleic acid to be analyzed is directly dripped by the dried film to be absorbed. Since the nucleic acid diffuses more rapidly under the dry condition of the film than in the wet condition, the nucleic acid to be analyzed can be quickly adsorbed by the film, thereby shortening the time required for the treatment of 11 200806799, and the equipment and solution required for the relevant steps. After the Langdings analysis nucleic acid is dropped on the 4 membrane, the j film can be dried by a method, and then the hybridization reaction can be carried out. Prior to the hetero-hexalysis reaction, the conventional analytical method requires (4) that the region on the film where the acid is not fixed is covered with a solution containing a blocking reagent to avoid future non-specific binding of the probe. However, in the method of the present invention, it is not necessary to carry out the aforementioned hybridization anti-X reaction, and the probe nucleic acid is directly added to the membrane for hybridization reaction, and the reaction can be completed only in a few minutes. In the case where the acid solution is added to the film in a dry form, the probe nucleic acid: liquid is rapidly attracted to the capillary phenomenon generated by the fine pores of the film, and is faster than the wet state of the white The film absorbs and enters the pores in the film. The nucleic acid in the probe nucleic acid solution moves in the film by Brownian motion and simultaneously searches for a thief to be analyzed. The DNA of 3 核核 mer mer mer mer mer mer mer mer mer mer mer mer mer mer mer For example, in the μπι film hole, the diffusion time is only 〇6 seconds, and the 5000-mer DNA is only 25 seconds. It is only less than 1 minute to complete the annealing time of the primer and the sample nucleic acid according to the well-known polymerase chain reaction (p〇lymerasechah PCR). Therefore, under the conditions of the present invention, the probe nucleic acid can complete the base pairing in a relatively short period of time, so that it takes ten hours to shorten the conventional hybridization reaction, and it is completed in a few minutes. On the other hand, as described above, the binding of the probe nucleic acid to the nucleic acid to be analyzed is the binding force of the hydrogen bond of the lower 7 (pUrine) or the densely bite (pyrimi), and the hydrogen bond is formed. It is quite rapid, and it is also faster than the adsorption rate of the probe nucleic acid to the film. The binding force between the nucleic acid and the nucleic acid to be analyzed is stronger than the adsorption between the probe nucleic acid and the film, and the adsorption between the nucleic acid and the film to be analyzed is The force of the needle also has a large adsorption capacity on the film, and the nucleic acid of the domain needle is trapped in the pore of the thin flag compared with the nucleic acid to be analyzed which is not like a macromolecule. Therefore, it can be easily washed away in the subsequent cleaning reaction. The probe nucleic acid which is non-specifically combined with the film exhibits a low background noise hybridization reaction, which is also the reason for the pre-hybridization blocking step, but to avoid the original analysis The nucleic acid is washed and washed, and the cleaning reaction Mxk is finely packed for 5 minutes after the red solution is washed, and the cap is preferably 4 to 6 minutes. 'Under the rinse reaction, the hybridization reaction film can be subjected to the next step. Father reaction shouted _. Hybrid The signal _ can be _ currently known as a kit, and can be quickly corrected by means of photodetection to within a few minutes. The photoelectric detection type can include: impedance detection, capacitance value detection, resistance value The system or electrochemical test is not limited to this. / / Zong Shang said 'by the rapid _ printing and analyzing method of the material, the benefits of the steps (1) to (3) of the nucleic acid treatment to be analyzed or steps: (5) The nucleic acid hybridization step is only necessary to spend a few minutes to add the above steps, and only takes a total of (five) 15 minutes. Compared with the traditional method, it is a significant and effective time and material for improving the nucleic acid printing analysis method. The following is a description of the embodiments of the present invention, and the following examples are set forth to illustrate the invention, and are not intended to limit the scope of the present invention. In the spirit and scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims. [Embodiment] Please refer to the second figure, which The flow chart of the embodiment of the present invention is an embodiment of the method for analyzing a rapid nucleic acid fragmentation method, which comprises the steps of analyzing the nucleic acid treatment S50, the nucleic acid hybridization reaction S6〇, and the detection of the hybridization message s4〇. The blind first is to be analyzed. The nucleic acid is transferred to the substrate, and the nuclear to be analyzed is fixed, and the money is directly subjected to the secret treatment step. The τ 隹 parent pairing reaction of the nucleic acid to be analyzed is analyzed. Finally, the hybridization signal is speculated, and the nucleic acid print can be invented. The step of the analysis of the collapse analysis. The second diagram of the analysis of the nucleic acid to be analyzed, the step of analyzing the nucleic acid to be analyzed, includes a substrate provided with a hole in S51; in the step, it is prepared in 52G. The nucleic acid to be analyzed is delivered to the substrate while the nucleic acid is adsorbed to the substrate; in step s53, the nucleic acid to be immobilized is immobilized on the substrate. The substrate in the step S51 may be a 'membrane membrane, a stone wafer, a glass, a magnetic bead or a metal particle, but is not limited thereto. Therefore, after the nucleic acid to be analyzed is transferred to the substrate, in step S52 and step S53, the nucleic acid to be analyzed can be rhyme on the silk by means of dry adsorption, electro-adsorption, magnetism, and the like.

不僅限於前沭。 J 14 200806799 取待分析核酸處理步驟S50完成後，進行核酸雜交反應為S6G。本發明實施例中之酸雜交反應步驟S6G並不包 $習知之前雜交步驟，亦即不利用阻斷劑將該基質上未固定有該待分析核酸區域加以阻斷。步驟S6〇，其包括步驟 S61，係將一含有探針核酸之溶液加入該基質上進行雜交反應’使該探針核酸與該絲上之騎分析核酸進行驗基 -對’以及步驟S62 ’將未與該待分析核酸相結合的該探針核酸分離去除。最後進行步驟S4Q，對雜交訊息進行侦測。资測的方式可包括··呈色反應檢測 S41、放射線曝光颂衫S42、冷光反應檢測S43、光電反應檢測以4，但並不僅限於此。 5月茶閱第二圖’該圖係本發明第二實施例之流程示意圖。本發明快速減印潰分析方法之實施例中，包括待分 ^核酸處if S7G、核酸雜交反應，與雜交訊息的侧S4〇等步驟。首先將將待分析核酸滴放於薄膜上，並進行待分析核酸的固定’而後不經前雜交步驟而直接進行待分析核酸之雜統對反應，最後_交訊號精_，即可完成本I明核I印心析之步驟流程。兹就各步驟以實施例說明實施方式如下：實施例一：待分析核酸處理待分析核酸步驟S70，其係於步驟S71中提供一可吸附待分析核酸之薄膜，而後進行步驟 S72，將步驟S720 200806799 中所製備之該待分析核酸滴放於該薄膜上，而後進行如步驟S731以乾燥方式或步驟S732以UV照射方式，將待分析核酸固定於薄膜上，步驟S732後尚可進一步經步驟 S73l，將薄膜加以乾燥。其亦可先經步驟S732以UV照射方式，再經步驟S731，將薄膜加以乾燥。，步驟S71中所提供之薄膜可為目前所常見之尼龍膜或硝化纖維紙’但並不以此為限。尼龍膜可帶有正電荷或不 T電’其可吸附之核酸可大於400 pg/cm2以上，而硝化纖維紙所得吸附之核酸則約在75至110 pg/cm2之間。尼龍膜與硝化纖維紙之孔洞直徑可以為〇·1 μιη至50 μπι之間’此孔徑可依據待分析核酸分子量大小而選擇適用之孔徑大小，核酸愈大時所使用之孔徑也愈大，其中以〇.2jLmi 與〇·45 μιη為較佳。Not limited to the front. J 14 200806799 After the completion of the analysis nucleic acid treatment step S50, the nucleic acid hybridization reaction is carried out as S6G. The acid hybridization reaction step S6G in the examples of the present invention does not involve the prior hybridization step, that is, the blocker is not immobilized with the nucleic acid region to be analyzed. Step S6〇, which comprises the step S61, adding a solution containing the probe nucleic acid to the substrate for hybridization reaction 'the base of the probe nucleic acid and the riding analysis nucleic acid on the silk for the base-pair' and the step S62' The probe nucleic acid that is not bound to the nucleic acid to be analyzed is separated and removed. Finally, step S4Q is performed to detect the hybridization message. The measurement method may include, for example, color reaction detection S41, radiation exposure, shirt S42, cold light reaction detection S43, and photoelectric reaction detection, 4, but not limited thereto. The fifth tea is read in the second drawing. This figure is a schematic flow chart of the second embodiment of the present invention. In the embodiment of the rapid de-inking analysis method of the present invention, the steps include: if S7G of the nucleic acid, nucleic acid hybridization reaction, and side S4〇 of the hybridization message. First, the nucleic acid to be analyzed is dripped on the film, and the nucleic acid to be analyzed is fixed, and then the hybrid reaction of the nucleic acid to be analyzed is directly performed without the pre-hybridization step, and finally, the I can be completed. The process of clearing the nuclear process. The embodiments are described as follows in the following steps: Example 1: The nucleic acid to be analyzed is subjected to the nucleic acid to be analyzed, step S70, in which a film capable of adsorbing the nucleic acid to be analyzed is provided in step S71, and then step S72 is performed, and step S720 is performed. The nucleic acid to be analyzed prepared in 200806799 is dropped on the film, and then the nucleic acid to be analyzed is fixed on the film by UV irradiation in step S731 or in step S732, and step S732 can be further followed by step S73l. , the film is dried. Alternatively, the film may be dried by UV irradiation in step S732 and then in step S731. The film provided in the step S71 may be the conventional nylon film or nitrocellulose paper', but is not limited thereto. The nylon membrane may be positively charged or not. The adsorbable nucleic acid may be greater than 400 pg/cm2, and the nitrocellulose may have an adsorbed nucleic acid of between about 75 and 110 pg/cm2. The diameter of the pores of the nylon membrane and the nitrocellulose paper can be between 1·1 μιη and 50 μπι. 'This pore size can be selected according to the molecular weight of the nucleic acid to be analyzed. The larger the nucleic acid is, the larger the pore diameter is. It is preferable to use 〇.2jLmi and 〇·45 μιη.

步驟S720中關於待分析核酸之製備，係利用習知核酸萃取與純化技術所取得。待分析核酸係等待確認該核酸上是否具有所需基因序列或DNA/RNA片段之核酸樣本，因此本發明得利用於檢測DNA或RNA。經萃取純化之 DNA可溶於ΤΕ之緩衝溶液（10mM Tris-Hcl，pH8 〇;丨mM EDTA)中，而RNA則可溶於去除核醣核酸酶之無菌水中備用。、岫述热淪是DNA或是RNA待測樣本，必須保持在單月又狀怨，始能與單股探針形成良好的配對，故一般習知方法皆須經過變性（Denature)步驟。若欲進行變性，對於 DNA樣本’可於前述製備之謝八溶液巾加人a」倍體積 16 200806799 之1 N氫氧化鈉（Na〇H)，於37°C下反應5分鐘後，置放於冰上’而後再加入1倍體積的2 Μ錯酸氨（ammoniinn acetate’pH 7.0)。RNA樣本之變性方式，則可於前述製備的RNA溶液中加入100%甲醯胺（formamide，終濃度為 50%)、37% 甲醛（formaidehyde，終濃度為 7%)與，20 倍SSC溶液（終濃度為1倍），而後於68°C下反應15分鐘，其後再置放於冰上，最後加入2倍體積的20倍SSc 溶液。然而，本發明發現，於步驟S731與步驟S732中，待为析樣本即得因為高溫之加熱或之照射而變性（參見後述）λ無庸預先進行變性步驟，故於本發明實施例中，並未有變性此過程，因而亦可減少此步驟所需之試劑、溶液與反應時間。待分析核酸製備完成之後，即可進行步驟S72，將該待分析核酸滴放於薄膜上。於本實施例中，可省略習知薄膜浸潤之過程’使薄默乾餘而直_放待分析核酸。其原因在於，核酸於薄膜乾餘況下可迅速為薄膜孔洞所產生之毛細現象所吸引滲透’故比於濕潤狀況下之盘吸附更為快速，因而可進—步縮短待分析核酸處理步驟 S70^t需之時間’以及該步驟所需之設備、溶液與試劑。，析核酸滴放的量可依據薄膜可以承置加以計算，-般可為〇·5至2咕咖2，較 -邊 cm2 ’但並不以此為限。、、、μ§ 經步驟S72後，即進行步驟S731，將薄膜予化，乾燥之方式可將該薄膜置於肋。^ ^ 王下（較佳者 17 200806799 為90°C至ll〇°C )，烘乾1至10分鐘，但並不限於此種方式。乾燥後之薄膜，待分析核酸已固定吸附於薄膜上，但為進一步強化待分析核酸之固&附著力，可進行步驟 S732，將該薄膜進一步置於2至5焦耳/cm2，波長254 rim 之紫外光下照射5至10分鐘。另外，亦可先進行步驟S732 之紫外光照射，再進行步驟S73l之乾燥化步驟。無論係經步驟S731或步驟S732，藉由高溫或紫外光之照射，皆可將待分析核酸加以變性，因而可省略前述將待分析核酸變性之步驟。本發明實施例中之待分析核酸處理步驟，，省略了習知薄膜浸潤步驟SU、變性待分析核酸步驟 S22以及烘乾固定待分析核酸步驟S24等步驟（請來閱第一圖），故可簡化核酸印潰分析所需之操作步驟，並同時減少該些步驟所需之試劑、溶液與反應時間。實施例二：核酸雜交反應 =待分析核酸⑽於_上後，接著進行核酸雜交反 =S8:。於一般情況下，由於薄膜上未固定有待分析仍可吸附核酸，若不將該些區域加以阻斷，則加八同為核酸之探針時，探舢薄膜、斗核k將非專一性地結合於 /寻联上，因此於習知技術中，推地必需#、# & 進仃雜交反應前係無可避免雜訊值Γ先進订刖雜父反應，以減低將來雜交訊號之背影、、而，由於本發卿交反麵，係先將㈣有待分析 200806799 核酸之薄膜加以乾燥，故再加人探針核酸溶液時，該探針核酸溶液得迅速為薄膜孔賴產生毛細現象之吸引力所吸入’吸人後再藉由布㈣動，即可於極短時間内尋找到可相配對之待分析減，並於數十科間喊成二者驗基間之氫鍵結合。驗基配對過程中，探針核酸與待分析核酸虱鍵結合之速度要比與薄膜快，且探針核酸並未經過待分析核酸之乾燥目定過程，因此騎_與待分析核酸之配對齡力要比探針猶與薄朗之吸附力強，而待分析核酸與薄膜間之吸附力亦較探針核酸對薄膜之吸附力為大’且探針核酸通常係較短小之分子，不似大分子的待分析，酸係卡陷在薄膜之孔洞中。因此，探針核酸不易附著於薄膜内轉分析減所未吸附之區域上，故於清洗反鹿中仍可輕易清洗掉與薄膜非專-性結合之探針核酸，而現^低背景雜訊之雜交反應結果。故於本發明實施例中，並無需進行前雜交反應，因而可再簡化此道習知技術中必要之操作步驟，並同時減少此步驟所需之試劑、溶液盘反應時間。 ^ 因此，於本發明中之核酸雜交反應S8〇流程中，係直接進行步驟S81之驗基配對反應，將所製備好標記有顯示分子的探針核酸，吸取適量滴放於薄膜上吸附有待分析核酸之表面，並於贼至7(rc下，反應2至5分鐘。其中，車乂么之反應溫度為46 C至56°C，但其得依探針核酸分子之Tw值加以調整，於此並未設有特別的限制。由於先前滴放待分__，薄難呈錢狀態，待分析核酸溶液 19 200806799 逮為薄膜所吸收，吸收後，薄臈再一次予以乾燥化，薄腹王針核酸溶液時，該探針核酸溶液亦可被迅速為動=見象所產生之吸引力吸入，吸入後再藉由布朗運部之=勒配對之待分析_。探針核酸進入薄膜内外六，除藉由自然毛細現象之負麼吸力外，亦可:施加利用壓力叙方錢探針核酸迅速進人薄膜内部。膜另面上抽以真空，利用真”形成之負舰該二，、/酸進入該基質内部’亦可於該基質所連接之一流道、以加壓方式賴探針賊推擠進人該基質内部。所述方式僅係例示，並不以此為限。The preparation of the nucleic acid to be analyzed in step S720 is carried out by a conventional nucleic acid extraction and purification technique. The nucleic acid to be analyzed is a nucleic acid sample waiting to confirm whether the nucleic acid has the desired gene sequence or DNA/RNA fragment, and thus the present invention is utilized for detecting DNA or RNA. The extracted and purified DNA can be dissolved in a buffer solution of hydrazine (10 mM Tris-Hcl, pH 8 〇; 丨 mM EDTA), and the RNA can be dissolved in sterile water for removing ribonuclease for use. The enthusiasm is that the DNA or RNA sample to be tested must be kept in a single month and resentment, and it can form a good pair with the single-strand probe. Therefore, the conventional methods must undergo a Denature step. If the denaturation is desired, the DNA sample can be placed in the above-mentioned Xie Ba solution towel with a volume of 16 200806799 of 1 N sodium hydroxide (Na〇H), reacted at 37 ° C for 5 minutes, and then placed. On ice, 1 volume of 2 ammonium acetate 'pH 7.0' was added. For the denaturation of RNA samples, 100% formamide (final concentration 50%), 37% formaldehyde (formaidehyde, final concentration 7%) and 20 times SSC solution can be added to the RNA solution prepared above. The final concentration was 1 time), and then reacted at 68 ° C for 15 minutes, and then placed on ice, and finally 2 volumes of 20 times SSc solution was added. However, the present inventors have found that in steps S731 and S732, the sample to be analyzed is denatured by heating or irradiation of high temperature (see later). λ is not required to perform the denaturation step in advance, and thus, in the embodiment of the present invention, This process is denatured and thus reduces the reagents, solutions and reaction times required for this step. After the preparation of the nucleic acid to be analyzed is completed, step S72 is performed, and the nucleic acid to be analyzed is dropped onto the film. In the present embodiment, the process of the conventional film infiltration can be omitted, and the micronucleus can be left to be analyzed. The reason is that the nucleic acid can quickly penetrate the capillary phenomenon generated by the film pore under the dry condition of the film, so the disk adsorption is faster than that under the wet condition, so that the nucleic acid processing step S70 can be further shortened. ^t time required' and the equipment, solutions and reagents required for this step. The amount of the nucleic acid to be dropped can be calculated according to the film, which can be calculated as 〇·5 to 2咕2, which is not limited to the side cm2. After the step S72, the step S731 is performed to pre-process the film, and the film can be placed in the rib by drying. ^ ^ Wang Xia (preferably 17 200806799 is 90 ° C to ll 〇 ° C), drying for 1 to 10 minutes, but is not limited to this method. After drying, the nucleic acid to be analyzed has been fixedly adsorbed on the film, but in order to further strengthen the solid & adhesion of the nucleic acid to be analyzed, step S732 may be performed, and the film is further placed at 2 to 5 Joules/cm2, and the wavelength is 254 rim. Irradiate for 5 to 10 minutes under ultraviolet light. Alternatively, the ultraviolet light irradiation in step S732 may be performed first, and the drying step in step S73l may be performed. The nucleic acid to be analyzed may be denatured by irradiation with high temperature or ultraviolet light, either by step S731 or step S732, and thus the aforementioned step of denaturation of the nucleic acid to be analyzed may be omitted. In the nucleic acid processing step to be analyzed in the embodiment of the present invention, the steps of the conventional film infiltration step SU, the denaturation of the nucleic acid to be analyzed step S22, and the step of drying the fixed nucleic acid to be analyzed step S24 are omitted (please refer to the first figure). Simplify the procedures required for nucleic acid blotting analysis while reducing the reagents, solutions and reaction times required for these steps. Example 2: Nucleic Acid Hybridization Reaction = After the nucleic acid (10) to be analyzed is on _, then nucleic acid hybridization is reversed = S8:. Under normal circumstances, since the nucleic acid is not immobilized on the film to be analyzed, if the region is not blocked, when the probe is a nucleic acid probe, the probe film and the core k will be non-specifically In combination with / search, in the conventional technology, push the necessary #, # & before the hybridization reaction is inevitable noise value, advanced ordering father reaction, to reduce the back of the future hybrid signal, However, since the hair of the hair is sent to the opposite side, the film of the 200806799 nucleic acid to be analyzed is first dried. Therefore, when the probe nucleic acid solution is added, the probe nucleic acid solution rapidly attracts the capillary phenomenon of the film pores. The force inhaled 'inhalation and then by cloth (four) to move, in a very short time to find a pair of pairs to be analyzed and reduced, and shouted into the hydrogen bond between the two groups. During the base pairing process, the probe nucleic acid binds to the nucleic acid to be analyzed at a faster rate than the membrane, and the probe nucleic acid does not pass through the drying process of the nucleic acid to be analyzed, so the riding age is matched with the nucleic acid to be analyzed. The force is stronger than the adsorption force of the probe, and the adsorption between the nucleic acid and the film to be analyzed is also larger than the adsorption capacity of the probe nucleic acid to the film, and the probe nucleic acid is usually a shorter molecule, which is not like For the macromolecule to be analyzed, the acid card is trapped in the pores of the film. Therefore, the probe nucleic acid is not easily attached to the untransferred region of the film, so that the probe nucleic acid which is non-specifically bound to the film can be easily washed away in the anti-deer, and the background noise is low. The result of the hybridization reaction. Therefore, in the embodiment of the present invention, the pre-hybridization reaction is not required, so that the necessary operation steps in the prior art can be further simplified, and at the same time, the reagent and solution plate reaction time required for the step can be reduced. Therefore, in the nucleic acid hybridization reaction S8〇 process of the present invention, the test pairing reaction of step S81 is directly performed, and the probe nucleic acid labeled with the display molecule is prepared, and an appropriate amount is dropped onto the film for adsorption to be analyzed. The surface of the nucleic acid, and the reaction is 2 to 5 minutes at the thief to 7 (rc). The reaction temperature of the ruthenium is 46 C to 56 ° C, but it is adjusted according to the Tw value of the probe nucleic acid molecule. There is no special restriction on this. Because the previous drop is to be divided into __, the thin is difficult to be in a state of money, the nucleic acid solution to be analyzed 19 200806799 is absorbed by the film, and after absorption, the thin sputum is once again dried, the thin belly king When the nucleic acid solution is needled, the probe nucleic acid solution can also be rapidly inhaled by the attractive force generated by the appearance, and then inhaled by the Brownian Department to be analyzed. The probe nucleic acid enters the inside and outside of the film. In addition to the negative suction caused by the natural capillary phenomenon, it can also be: applying the pressure of the probe to the nucleic acid to quickly enter the inside of the film. The vacuum is applied to the other side of the membrane, and the negative ship formed by the true one is used. /acid enters the interior of the matrix' The probe can also be pushed into the interior of the substrate by a flow path in a flow path connected to the substrate. The manner is merely illustrative and not limited thereto.

依擴散時間⑺與擴散距離⑷、擴散係數（D)間之關係·· T = d2/D，長度為30個核普酸（3〇-mer)之DNA (擴散係數為4 X 1(T" m2/sec)，若於5· _薄膜孔洞中移動其擴放日守間僅需〇·6秒，而5000_mer之DNA (擴散係數為lxl(T12m2/sec)亦僅需25秒。因此，在不到卜分鐘之時間内，分佈於薄膜孔洞内之探針核酸，即可在各該孔洞内完成其鹼基配對過程。藉此，即可縮短原習知探針核酸於薄膜濕潤狀況下由薄膜外緩慢擴散至薄膜表面，再由薄膜表面擴散移動至薄膜内所需漫長之時間，使探針核酸迅速進入薄膜纖維中，並迅地完成雜交配對，因此可^ 幅縮短雜交反應時間，並使步驟S81於數分鐘内即可完成。經步驟S81之鹼基配對反應後，則是進行清洗反應步驟S82，將未配對的探針核酸沖洗掉。如前所述，探針核酸與待分析核酸之配對係相當迅速，故該探針核酸並不易 20 200806799 ，著於薄膜上，且探針賊並未經分_酸之乾燥固疋過耘，因此探針核酸與待分析核酸之配對結合力要比針核酸與薄顯之吸附力強，故於清洗反應巾，可輕易、、主洗掉與薄膜非專-性結合之探針核酸，而呈現出低背景^ 訊之雜交反應結果。但為戦同時清洗掉待分析核酸/，、清洗反應時以低離子強度之缓娜液進行漂洗為較佳/且^ 洗的㈣僅需數分鐘即可，—般係3至7分鐘，較佳者為 4至6分鐘。所述低離子強度之緩衝溶液，可利用含有0·05 至W5-倍之SSC溶液，於室溫下漂洗4至6分鐘，共漂洗二次。若係利用消化纖維紙，則可進一步加入〇〇5^ 〇^5/(w/v)之SDS進行漂洗。其巾，ssc溶液較佳濃度為0·1倍，而SDS溶液較佳濃度為〇1%(w/v)。疋故本發明貫施例步驟S8〇之核酸雜交反應中，省 t有習知前雜交反應S31(請參閱第一圖），並將雜交反應了間由十數小時縮減至數分鐘，_也將原清洗反應⑶ 所而之日守間鈿短為數分鐘，故於本發明步驟％〇中，亦是大幅降低所需反應時間以及相關試劑、溶液之成本支出。 “由上述可知，藉由本發明之快速核酸印潰分析方法，無論係待分析核酸處理_或是核酸雜交反應s8〇等步 =皆僅需花費數分鐘之時間。加總上述步驟，也總共僅而化費3G至45分鐘，相較於傳統需要至少二天之方法，係大巾田並有效改善核酸印潰分析方法所需之時間與材料成本。 200806799 前述實施例中先將待分析核酸傳遞（滴放）並固定至基質（薄膜）’而後再加人核酸探針進行雜交反應之方式，亦可依據不同之顧目的，改變為先將探針減傳遞（滴放）並固定至基質（薄膜）上後，再加人待分析核酸進行雜交反應。由於-般所謂探針核酸，係標記有榮光、皇色等::示ί仅滅W，11此，若縣賴針滅附於基貝（薄膜）上，雜父反應後所有探針核酸仍然吸附於基貝（薄膜）上’-般呈色、螢光、放射線曝光顯影等指示型探針與偵測方法將因無法區別而不能翻，此時可利用阻抗檢測、電容值檢測、電阻值檢測或電化學檢測等方式加以彳貞測。經漂洗後之雜交反應薄膜上只剩能夠與待分析核酸相配對的探針核酸，此時即可進行步驟S4〇關於雜交反應几號之偵測。進行雜父反應訊號彳貞測時，可配合探針所標圮之顯示分子，而使用適當的檢測方式。關於檢測方式，目前市面上有相當多的反應組套，除可利用該組套進行呈色或其他顯示反應外，該組套同時亦包括有製備探針所需之試劑與溶液。在呈色反應檢測S41方面，目前最常見的有利用DIG (digoxigenin)的呈色系統，其通常係藉由一連接有驗性去磷酸酶（alkaline phosphate)之anti-DIG抗體與氮藍四嗤（nitrobluetetrazolium，簡稱 NBT)、漠-4-氣-3-口引哚基磷酸（5_bromo冰chloro_3_indolyl phosphate，簡稱BCIP)或CSPD@等呈色受質進行呈色反應。熟習本發明技術領域之技藝者根據本說明書之敘述亦可了解，探 22 200806799 針核^製備時亦可以生物素（bi〇tin)來標記，之後於呈色反應中再藉由卵白素（streptavidin)與酵素進行呈色反應。 ^ ’亦可利用標記以放射線元素32p或％之放射二 j測方式S42，或是湘HEX、Cy3及印#冷光反應才双測方式S43。所述檢測方式僅係例示，其他例如：阻抗 1測、電容值檢測、電阻值檢測、電化學檢測等光電反應板測S44方式，以及質量檢測或重量檢測方式亦皆可利用’但並不僅限於此。實施例三：核酸烘乾固定時間分析準備一呈乾燥狀之尼龍膜，其孔徑大小為〇·45 pm，利用活頁紙穿孔機，於前述尼龍膜上，壓製出直徑ό mm (面積約28 mm2)的圓形尼龍膜片。之後，於每一尼龍膜片正中央處直接滴放3 μΐ 1〇〇 pm〇l的待分析核酸溶液（同時滴放有一 3 μΐ去離子水作為控制組），再將此些尼龍膜片置於加熱器上，於100艺溫度下，以不同時間進行烘烤，藉以分析待分析核酸於不同固定時間下之固定差異性。將固定以待分析核酸之尼龍膜置於低離子強度缓衝 /谷液（如0.1倍SSC+0.1% (w/v)之SDS)中進行漂洗，以洗去未固定之待分析核酸，漂洗時每次10分鐘，共五次。之後，於每一尼龍膜片正中央處再滴放3μ1100ριη〇1 標記有螢光分子的探針核酸溶液，以46°C進行鹼基配對 1〇分鐘後’以緩衝溶液（〇丨倍ssc+〇1% (w/v)之SDS) 23 200806799 進行漂洗，漂洗時每次ίο分鐘，共三次。最後分別對不同固定時間之尼龍膜，偵測分析其螢光值。請參閱第四圖，該圖係本發明以不同時間進行核酸固定之比較結果圖。圖中，橫座標係烘乾固定時間，縱座標係螢光訊號強度。由圖中可明顯看出，烘乾固定時間:約於 30秒至1分鐘以上時，螢光訊號相對於控制組即有相當的差異性，因此於本發明中，烘乾固定之時間約於30秒至1 分鐘即可完成。貫施例四：驗基配對時間分析正放有準備一呈乾燥狀之尼龍膜，其孔徑大小為0.45 μηι，利用活頁紙穿孔機，於前述尼龍膜上，壓製出直徑6 mm (面積約28 mm2)的圓形尼龍臈片。之後，於每一尼龍膜片中央處直接滴放3 μΐ 100 ng的待分析核酸溶液（同時滴、〇 W去难子水作為控制組），再將此些尼龍膜片置尸上’於10(TC溫度下供烤10分鐘。之後，於每— =¾片正中央處再滴放3 μΙ刚pmQl標記有螢光分子 ^針核酸溶液’以贼於不同時間下進行驗基配對，並洗溶液倍ssc+ G 1% (w/v)之sds)進行漂配對^時每f 1G分鐘’共三次。最後分別對不同驗^ 守間之尼龍膜，偵測分析其螢光值。明參閱第五圖，該圖係本發明以不同時間進行驗基配 24 200806799 對之比較結果圖。圖中，橫座標係驗基配對之反應時間，縱座標係螢光訊號強度。由圖中可明顯看出，鹼基配對時間約於2分鐘以上時’螢光訊號相對於控制組即有相當的差異性，因此於本發明中，驗基配對所需時間約2分鐘即可完成。實施例五：以微流體晶片進行核酸印潰分析、準備一微流體晶片（Microfluidics)，該微流體晶片内已裝设有一孔徑大小為〇·45 μιη，尺寸為4 χ 7mm之尼龍膜。以注射筒連接一連接管分別注入10 μΐ ( 10 ng)五· 之D1^A (對照組）與五· Mr而之DNA (實驗組）以及10 μ1 之去離子水（控制組），並使該些待分析核酸溶液與去離子水引流至尼顏上為其所吸附。之後，將職流體晶片置於加熱ϋ上，以12(rc烘乾1〇分鐘，使待分析核酸固定於尼龍膜上。狄、j後進行核酸雜交反應’將1〇μ1 (10Ρ_)標記有筆ίίΤ之探針核酸（該核酸係僅可與瓦—己對）埶。ί爪遏内加壓〉主入尼龍膜後，再將該微流體晶片置於加以价奸較反應2分鐘。錢接續注入清洗二^/.1倍SSC+ G.1% (w/v)之SDS)進行漂洗，漂洗二Γ1爪卜2分鐘，共四次。每漂洗1次，記錄其螢光偵測值。 25 200806799 請參閱第六圖，該圖係本實施例以微流體晶片進行本發明核酸印潰分析方法之雜交結果圖。圖中，橫座標係漂洗次數，縱座標係螢光訊號強度。「♦」係指五· C0/z.iDNA，」係4日僅有棟針核酸之控制組’「·」係指五纟似士之 DNA。由圖中可發現，隨著漂洗次數之增加，螢光訊:號也愈低，其係因探針核酸逐漸被漂洗去除所致。另外，由於才米針核酸無法與五· co//之DNA配對，因此所能價測到之螢光訊號也最低。而其訊號強度高過單純只有探針核酸控制組之原因在於，五· C0//DNA係先附著於尼龍膜上，並填充於尼龍膜之孔洞中，因此後來所加入屬於背景值的探針核酸就較不易附著於尼龍膜上。相對之下，未附著有待分析核酸之控制組上，探針核酸可隨意附著，因此附著之比例較多，背景值之強度因而也較高。但從結果可知，約在弟2次漂洗後’即可區分出待分析核酸是否為探針核萨戶斤得配對，而在第3次漂洗後，即明顯可以區分。【圖式簡單說明】第一圖係習知核酸印潰分析方法之流程示意圖。第二圖係本發明實施例之流程示意圖。第三圖係本發明第二實施例之流程示意圖。第四圖係本發明以不同時間進行核酸固定之比較結果圖。圖中，橫座標係烘乾固定時間，縱座標係營光訊號強度。 26 200806799 第五圖係本發明以不同時間進行鹼基配對之比較結果圖。圖中，橫座標係驗基配對之反應時間，縱座標係螢光訊號強度。第六圖係以微流體晶片進行本發明核酸印潰分析方法之雜交結果圖。圖中，橫座標係漂洗次數，蜂座標係螢光訊號強度。♦:五.c〇//DNA ; :控制組； •:五.DNA。【主要元件符號說明】 S10 薄膜前處理 S11 提供一薄膜 S12 浸潤該薄膜 S20 待分析核酸處理 S21 製備待分析核酸 S22 變性該待分析核酸 S23 滴放待分析核酸於該薄膜上 S24 烘乾固定該待分析核酸 S25 UV固定該待分析核酸 S30 核酸雜交反應 S31 前雜交反應 S32 加入探針核酸，進行雜交反應16小時以上 S33 清洗反應 27 200806799 S40 雜交訊息的偵測 S41 呈色反應檢測 S42 放射線曝光顯影 S43 冷光反應檢測 S44 光電反應檢測 / S50 待分析核酸處理 S51 提供一基質，該基質係具有孔洞 S520 _製備待分析核酸 ~ S52 傳遞該待分析核酸於該基質上 S53 將該待分析核酸固定於該基質上 S60 核酸雜交反應 S61 加入探針核酸，進行驗基配對反應 S62 將未與該待分析核酸相結合的探針核酸分離去除 S70 待分析核酸處理 S71 提供一薄膜 S720 製備待分析核酸 S72 滴放該待分析核酸於該薄膜上 S731 乾燥該薄膜 S732 UV固定該待分析核酸 S80 核酸雜交反應 S81 加入探針核酸，進行鹼基配對反應數分鐘 28 200806799 S 8 2 以缓衝溶液進行清洗反應 29According to the relationship between diffusion time (7) and diffusion distance (4), diffusion coefficient (D) · T = d2/D, the length of 30 nucleotides (3〇-mer) DNA (diffusion coefficient is 4 X 1 (T" M2/sec), if it is moved in the 5· _ film hole, it only takes 〇6 seconds to move the daytime, and the DNA of 5000_mer (the diffusion coefficient is lxl (T12m2/sec) takes only 25 seconds. Therefore, In less than a minute, the probe nucleic acid distributed in the pores of the membrane can complete the base pairing process in each of the pores, thereby shortening the original probe nucleic acid in the wet state of the membrane. The film diffuses slowly to the surface of the film, and then the diffusion of the film surface into the film takes a long time, so that the probe nucleic acid rapidly enters the film fiber and rapidly completes the hybridization pairing, thereby shortening the hybridization reaction time, and Step S81 can be completed in a few minutes. After the base pairing reaction in step S81, the washing reaction step S82 is performed, and the unpaired probe nucleic acid is washed away. As described above, the probe nucleic acid is to be analyzed. The pairing of nucleic acids is quite rapid, so the probe nucleic acid is not easy to 20 200806799, on the film, and the probe thief is not dried and separated by _ acid, so the binding ability of the probe nucleic acid to the nucleic acid to be analyzed is stronger than that of the needle nucleic acid and the thin display. The reaction towel can be cleaned, and the probe nucleic acid which is non-specifically combined with the film can be washed off easily, and the hybridization reaction result of low background is presented, but the nucleic acid to be analyzed is washed at the same time, and the reaction is cleaned. Rinsing with a low ionic strength of the solution is preferred/washed (4) in just a few minutes, typically 3 to 7 minutes, preferably 4 to 6 minutes. The low ionic strength buffer The solution can be rinsed at room temperature for 4 to 6 minutes with a SCP solution containing 0.05 to W5-fold, and rinsed twice. If the digested fiber paper is used, 〇〇5^ 〇^5/ can be further added. The (w/v) SDS is rinsed. The towel has a preferred concentration of ssc solution of 0.1 times, and the preferred concentration of the SDS solution is 〇1% (w/v). Therefore, step S8 of the present invention is carried out. In the nucleic acid hybridization reaction, there is a conventional pre-hybridization reaction S31 (see the first figure), and the hybridization reaction is reduced by ten hours. By reducing it to a few minutes, _ also shortens the day of the original cleaning reaction (3) to a few minutes, so in the step % of the present invention, the required reaction time and the cost of the relevant reagents and solutions are also greatly reduced. It can be seen from the above that, by the rapid nucleic acid fragmentation analysis method of the present invention, it takes only a few minutes for the nucleic acid treatment to be analyzed or the nucleic acid hybridization reaction to be s8, etc. The total of the above steps is only total. The cost of 3G to 45 minutes is compared with the traditional method of at least two days, which is a large towel field and effectively improves the time and material cost required for the nucleic acid fragmentation analysis method. 200806799 In the foregoing embodiment, the nucleic acid to be analyzed is first transferred (drip And fixing to the substrate (film) and then adding a nucleic acid probe for hybridization, or depending on the purpose, changing the probe to the substrate (drop) and fixing it to the substrate (film) After that, the nucleic acid to be analyzed is further added for hybridization reaction. Because of the so-called probe nucleic acid, the label is marked with glory, royal color, etc.:: ί only kills W, 11 this, if the county needle is attached to the kebe (film), all probe nucleic acids after the hetero-parent reaction still The probes and detection methods such as 'normal color, fluorescence, radiation exposure and development, which are adsorbed on the base film (film), cannot be turned over because they cannot be distinguished. In this case, impedance detection, capacitance value detection, and resistance value can be utilized. Detection or electrochemical detection, etc. Only the probe nucleic acid capable of pairing with the nucleic acid to be analyzed is left on the hybridization reaction film after the rinsing, and the detection of the number of the hybridization reaction in step S4 can be performed. When performing the detection of the heterogeneous reaction signal, the display molecule labeled with the probe can be used, and an appropriate detection method can be used. Regarding the detection method, there are a large number of reaction sets currently available on the market. In addition to the color or other display reactions that can be used for the detection, the set also includes the reagents and solutions required for preparing the probe. In terms of color reaction detection S41, the most common coloring system using DIG (digoxigenin) is usually an anti-DIG antibody linked to an alkaline phosphate and nitrogen blue tetramine. (nitrobluetetrazolium, abbreviated as NBT), desert-4-gas-3-mercaptophosphoric acid (5_bromo ice chloro_3_indolyl phosphate, referred to as BCIP) or CSPD@, etc. Those skilled in the art of the present invention can also understand from the description of the present specification that the detection of 22 200806799 can also be labeled with biotin (bi〇tin), and then by the white pigment in the color reaction (streptavidin). ) A color reaction with the enzyme. ^ ' can also use the mark with the radiation element 32p or % of the radiation measurement method S42, or Hunan HEX, Cy3 and India # luminescence reaction only double test mode S43. The detection method is merely an example, and other methods such as impedance 1 measurement, capacitance value detection, resistance value detection, electrochemical detection, etc., and the quality detection or weight detection methods are also available 'but not limited to this. Example 3: Nucleic Acid Drying Fixation Time Analysis A dry nylon membrane having a pore size of 〇·45 pm was prepared by using a loose-leaf paper punching machine to produce a diameter of ό mm (area of about 28 mm 2 ) on the aforementioned nylon membrane. Round nylon diaphragm. Then, directly deposit 3 μΐ1〇〇pm〇l of the nucleic acid solution to be analyzed in the center of each nylon membrane (at the same time, drop 3 μΐ of deionized water as a control group), and then place the nylon membranes. On the heater, at a temperature of 100 art, baking is performed at different times to analyze the fixed difference of the nucleic acid to be analyzed at different fixed times. The nylon membrane immobilized with the nucleic acid to be analyzed is subjected to rinsing in a low ionic strength buffer/glutle solution (for example, 0.1 times SSC + 0.1% (w/v) SDS) to wash away the unfixed nucleic acid to be analyzed, and rinsed. Each time 10 minutes, a total of five times. Thereafter, a probe solution of 3 μl 100 ι 〇 1 labeled with a fluorescent molecule was further dropped at the center of each nylon membrane, and base pairing was carried out at 46 ° C for 1 〇 after the buffer solution (〇丨 ssc + 〇 1% (w/v) SDS) 23 200806799 Rinse and rinse three times each time for ίο minutes. Finally, the nylon membranes of different fixed time were detected and analyzed for their fluorescence values. Please refer to the fourth figure, which is a comparison result of nucleic acid fixation at different times in the present invention. In the figure, the abscissa is the drying time, and the ordinate is the fluorescence signal intensity. It can be clearly seen from the figure that the drying fixed time: about 30 seconds to more than 1 minute, the fluorescent signal is quite different from the control group, so in the present invention, the drying and fixing time is about It can be completed in 30 seconds to 1 minute. Example 4: The base pairing time analysis is prepared by preparing a dry nylon membrane with a pore size of 0.45 μηι, using a loose-leaf paper punching machine to press a diameter of 6 mm on the aforementioned nylon membrane (area about 28 Round nylon cymbal of mm2). Then, directly deposit 3 μΐ 100 ng of the nucleic acid solution to be analyzed at the center of each nylon membrane (while dropping, 〇W to the water of the hard water as a control group), and then place the nylon membrane on the body. (Take for 10 minutes at TC temperature. Then, add 3 μΙ just pmQl labeled with fluorescent molecule ^Nucleic acid solution at the center of each -3⁄4 piece to perform pedigree pairing at different times and wash The solution times ssc + G 1% (w/v) of sds) was subjected to a drift pairing ^ three times every f 1G minutes. Finally, the nylon membranes of different inspections were detected and analyzed for their fluorescence values. Referring to the fifth figure, the figure is a comparison chart of the results of the present invention at different times. In the figure, the transverse coordinate system is the reaction time of the base pairing, and the ordinate is the fluorescence signal intensity. As is apparent from the figure, when the base pairing time is about 2 minutes or more, the fluorescent signal is quite different from the control group. Therefore, in the present invention, the time required for the base pairing can be about 2 minutes. carry out. Example 5: Nucleic acid chip analysis was performed on a microfluidic wafer, and a microfluidic chip having a nylon membrane having a pore size of 〇·45 μm and a size of 4 χ 7 mm was prepared. Connect a connecting tube with a syringe to inject 10 μΐ (10 ng) of D1^A (control) and V. Mr. DNA (experimental group) and 10 μl of deionized water (control group), and The nucleic acid solution to be analyzed and the deionized water are drained to the Nye for adsorption. After that, the working fluid wafer is placed on a heating crucible, and the nucleic acid to be analyzed is fixed on the nylon membrane by 12 (rc drying for 1 minute. The nucleic acid hybridization reaction after Di, j is marked with 1〇μ1 (10Ρ_)) Pen ίίΤ's probe nucleic acid (the nucleic acid system can only be paired with watts - )) ί ί 遏遏〉〉〉〉主主主主主主主主主主主主主主主主主主主主主主主主主主主主Continue to inject and clean 2/.1 times SSC+ G.1% (w/v) SDS) for rinsing, rinse 2 Γ 1 claw for 2 minutes, a total of four times. Each time it is rinsed once, the fluorescence detection value is recorded. 25 200806799 Please refer to the sixth figure, which is a hybridization result diagram of the nucleic acid fragmentation analysis method of the present invention using a microfluidic wafer in the present embodiment. In the figure, the abscissa is the number of rinses, and the ordinate is the intensity of the fluorescent signal. "♦" refers to the five C0/z.iDNA, which is the control group of only the needle nucleic acid on the 4th day. It can be seen from the figure that as the number of rinses increases, the lower the fluorescence signal: the lower the probe nucleic acid is gradually removed by rinsing. In addition, since the rice needle nucleic acid cannot be paired with the DNA of the five co//, the fluorescent signal detected by the price is also the lowest. The reason why the signal intensity is higher than the probe-only nucleic acid control group is that the C·C0//DNA is first attached to the nylon membrane and filled in the pores of the nylon membrane, so the probe belonging to the background value is added later. Nucleic acids are less likely to adhere to nylon membranes. In contrast, on the control group to which the nucleic acid to be analyzed is not attached, the probe nucleic acid can be attached at will, so that the ratio of adhesion is large, and the intensity of the background value is also high. However, it can be seen from the results that after about 2 rinses, it is possible to distinguish whether the nucleic acid to be analyzed is a pair of probes, and after the third rinse, it is clearly distinguishable. [Simple Description of the Drawing] The first figure is a schematic flow chart of a conventional nucleic acid printing and analyzing method. The second figure is a schematic flow chart of an embodiment of the present invention. The third figure is a schematic flow chart of the second embodiment of the present invention. The fourth panel is a comparison of the results of nucleic acid immobilization at different times in the present invention. In the figure, the abscissa is the drying time and the ordinate is the intensity of the optical signal. 26 200806799 The fifth figure is a comparison result of base pairing at different times in the present invention. In the figure, the transverse coordinate system is the response time of the pairing, and the ordinate is the fluorescence signal intensity. The sixth graph is a hybridization result diagram of the nucleic acid blot analysis method of the present invention using a microfluidic wafer. In the figure, the abscissa is the number of rinsing times, and the humbucker is the intensity of the fluorescent signal. ♦: c.c〇//DNA; : control group; •: five. DNA. [Major component symbol description] S10 film pretreatment S11 provides a film S12 infiltration of the film S20 nucleic acid treatment to be analyzed S21 preparation of nucleic acid to be analyzed S22 denaturation of the nucleic acid to be analyzed S23 Drip the nucleic acid to be analyzed on the film S24 drying and fixing Analytical nucleic acid S25 UV immobilization of the nucleic acid to be analyzed S30 nucleic acid hybridization reaction S31 pre-hybridization reaction S32 Adding probe nucleic acid, performing hybridization reaction for more than 16 hours S33 cleaning reaction 27 200806799 S40 detection of hybridization information S41 color reaction detection S42 radiation exposure development S43 Cold light reaction detection S44 Photoelectrochemical reaction detection / S50 The nucleic acid treatment to be analyzed S51 provides a matrix with a pore S520_Preparation of the nucleic acid to be analyzed~S52 Transfer the nucleic acid to be analyzed on the substrate S53 Fix the nucleic acid to be analyzed to the substrate The S60 nucleic acid hybridization reaction S61 is added to the probe nucleic acid to perform the base pairing reaction S62. The probe nucleic acid not bound to the nucleic acid to be analyzed is separated and removed. S70 The nucleic acid to be analyzed is treated. S71 provides a thin film S720. Preparation of the nucleic acid to be analyzed S72. The nucleic acid to be analyzed S731 is dried on the film S732 UV immobilized nucleic acid to be analyzed S80 nucleic acid hybridization reaction S81 is added to the probe nucleic acid to perform base pairing reaction for several minutes 28 200806799 S 8 2 Washing reaction with buffer solution 29

Claims

200806799 X. Patent application scope: The rapid nucleic acid crushing analysis method of the bbeking step includes the following steps: (1) providing a matrix having pores; (2) going-to-analyze Wei Transferred to the substrate, and the nucleic acid to be analyzed is adsorbed by the matrix; (3) the nucleic acid to be analyzed is immobilized on the substrate; (4) the nucleic acid to be analyzed is not fixed on the silk Where the region is blocked, a solution containing the probe nucleic acid is added to the substrate of step (3), and the probe nucleic acid is subjected to base pairing with the core I to be analyzed on the substrate (basepairing) a few minutes; (5) separating and removing the probe nucleic acid which is not combined with the nucleic acid to be analyzed in the step (4); and (6) detecting the hybridization message on the substrate via the step (5). 2. The rapid nucleic acid fragmentation analysis method according to claim 1, wherein the substrate in the step (1) is dry. 3. The rapid nucleic acid fragmentation analysis method according to claim 1, wherein the substrate in the step (1) is a membrane. 4. The method of claim 1, wherein the substrate is a nylon membrane. 5. The rapid nucleic acid fragmentation analysis method described in claim 3, wherein the matrix is nitr〇cenui〇se membrane in 200806799. 6. A method of rapid nucleic acid blotting analysis as described in claim 3, 4 or 5 wherein the pore diameter of the substrate is "〇" to 5 pm. 7. The rapid nucleic acid fragmentation analysis method according to claim 1, wherein the nucleic acid to be analyzed in the step (2) is deoxyribonucleic acid. 8. The method of claim 1, wherein the nucleic acid to be analyzed is rib〇nudeic acid. 9. The rapid capture nucleic acid crushing analysis method according to the application scope of the patent scope, wherein the step (3) is to dry the substrate at a temperature of 80 to 13 ', by which the nucleic acid to be analyzed is used. Fixed to the substrate. The rapid nucleic acid fragmentation analysis method according to claim 9, wherein the drying time of the step (3) is 1 minute. The rapid nucleic acid fragmentation analysis method according to the first aspect of the invention, wherein the substrate (3) is irradiated with ultraviolet light to fix the nucleic acid to be analyzed on the substrate. 12. If the scope of the patent application is 快速11 ί, the method of rapid nucleic acid fragmentation analysis is described; after V (3), the substrate to which the nucleic acid to be analyzed is immobilized is further dried. 13 f t | Gray, as in the fast-detecting nucleic acid fragmentation analysis method described in claim 1, the base pairing reaction time of the step (4) in 31 200806799 is 2 to 5 minutes. 14. The rapid nucleic acid fragmentation analysis method according to claim 1, wherein the step (5) is performed by rinsing with a low ionic strength buffer solution. 15. The rapid nucleic acid fragmentation analysis method according to claim 14, wherein the low ionic strength buffer solution in step (5) comprises a standard sodium citrate of 5 times the standard sodium citrate. , SSC). 16. The method of claim 1, wherein the low ionic strength buffer solution further comprises 〇〇 5% to 〇· 15 % (w/v) of sulfuric acid in the step (5).十二〇〇 C C C 溶液、、、、、、、、、、快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速快速The time is 3 minutes. 18. A method for rapid nucleic acid blotting analysis comprising the steps of: (1) k providing a substrate having a pore and being dried; (7) transferring the nucleic acid to be analyzed to the substrate, and causing the nucleic acid to be analyzed Adsorbing the substrate; (3) immobilizing the nucleic acid to be analyzed on the substrate; (Sentence - the solution containing the probe nucleic acid interacts with the substrate of step (3) at 4 ° C to 7 〇 (: 'Put the probe nucleic acid with the nucleic acid to be analyzed on the substrate for a few minutes; / 32 200806799 (5) The substrate of step (4) is rinsed with a buffer solution, so that it is not analyzed Nucleic acid phase binding _ probe nucleic acid separation and removal; and (6) detection of the hybridization message on the substrate according to step (5). 19, such as _ please go through the method of rapid capture nucleic acid analysis described in Item 18, The substrate in the step (1) is a film, and the method of analyzing the nucleic acid in the range of 19th, wherein the film is a nylon film, as described in claim 19 Rapid nucleic acid blotting analysis method, wherein the film is a 22, the patent of the patent range lS, N, 2 or U of the rapid nucleic acid printed mussel method of the method of 'the hole diameter of the matrix is 0.1 to 50 μπι. Women's claim patent scope item 18 The rapid nucleic acid fragmentation analysis method, the nucleic acid to be analyzed in the VIII (2) is the deoxyribonucleic acid. 4 The rapid nucleic acid fragmentation analysis method described in claim 18, wherein the step ( 2) The shaft nucleic acid to be analyzed is to be analyzed. 25. As described in the patent application 帛18, the rapid nucleic acid fragmentation analysis method, ', middle step' (3) is to place the substrate at 80 ° C to 13 Dry at 〇 ° C for 1 to 1 〇 minutes. 6 As described in the patent application 帛 18 J staff, the method of rapid nucleic acid blotting analysis, wherein step (3) is to irradiate the substrate with ultraviolet light for 5 to 1 〇. The rapid nucleic acid fragmentation analysis method described in claim 26 of the patent application scope, 33 200806799 further, the substrate is dried after the step (3). 28. The fast as described in claim 18 Nucleic acid blotting analysis method, wherein the base pairing reaction in step (4) The interval is 2 to 5 minutes. 29. The method for rapid nucleic acid fragmentation analysis described in the patent scope ,18, wherein the buffer solution in the step (5) comprises 〇·〇5 to 〇15 times the standard: the lemon 30. The rapid nucleic acid fragmentation analysis method according to claim 29, wherein the buffer solution further comprises 〇〇5% to 〇15% (w/v) sulfuric acid in the step (5). Sodium dodecate solution. 3 For example, please refer to the rapid nucleic acid fragmentation analysis method described in Item I8, wherein the rinsing time of the buffer solution in the step (5) is 3 to 6 minutes. 32. A method for rapid nucleic acid fragmentation analysis comprising the steps of: (1) providing a matrix having pores and being dried; (2) delivering a probe nucleic acid to the substrate to render the probe nucleic acid Adsorbing for the substrate; 1 (3) immobilizing the probe nucleic acid on the substrate; (4) interacting a solution containing the nucleic acid to be analyzed with the substrate of step (3) at 40 C to 70 C Having the probe nucleic acid paired with the nucleic acid to be analyzed on the substrate for several minutes; (5) rinsing the substrate of step (4) with a buffer solution, thereby not being associated with the probe nucleic acid The bound nucleic acid to be analyzed is separated and removed; and 34 200806799 (6) detecting the hybridization message on the substrate via step (5). 33. A rapid nucleic acid imprinting as described in claim 32, wherein the matrix is a film. Go to '34. As described in claim 33, the rapid subtraction of the film is a nylon film. Production, 35, as described in the patent application scope of the 33rd paragraph of the rapid nucleic acid printing 分八八八八 1 1 (1) in the film is a; 5 Xiaohua fiber paper. 36. The fast nucleic acid flash analysis method according to claim 32, 33, 34 or % of the patent application, wherein the pore diameter of the matrix is 〇1 to % sec. P. The rapid nucleic acid fragmentation analysis method according to claim 32, wherein the step (3) is to place the substrate at 8 ° C to 13 ° C, and twist to a minute. The main method of rapid nucleic acid fragmentation as described in claim 32, wherein step (3) is to irradiate the substrate with ultraviolet light for 5 to 1 minute. 39. A method for rapid nucleic acid fragmentation analysis according to claim 38 of the patent application, ν Ή 3), which is followed by stepwise stepping so that the substrate in which the probe nucleic acid is gj is dry. 4. The rapid nucleic acid fragmentation analysis method described in claim 32, wherein the reaction time of the step (4) is 2 to $ minutes. 41. The method as claimed in claim 32, wherein the buffer solution comprises 〇〇5 to 〇15 times standard lemon 35 200806799 sodium solution in step (5). 42 43 44 45 46 , such as the rapid nucleic acid fragmentation analysis described in the scope of patent patent, wherein the buffer solution in step (5) comprises 〇〇 5% to (w / v) sodium lauryl sulfate Solution. 4. The rapid nucleic acid fragmentation analysis method according to claim 32, wherein the step (5) rinses the buffer solution for 3 to 6 minutes. /, a rapid nucleic acid fragmentation analysis method, comprising the following steps: (1) providing a matrix having pores; (7) transferring the nucleic acid to be analyzed to the substrate, allowing the woman to analyze the adsorption of the matrix; 3) immobilizing the nucleic acid to be analyzed on the substrate and allowing it to be dry; (4) interacting a solution containing the probe nucleic acid with the substrate of step (3) at a milk C to 70 C, The probe nucleic acid is base paired with the nucleic acid to be analyzed on the substrate for several minutes; °X (5) the substrate of step (4) is rinsed with a buffer solution, thereby not combining with the nucleic acid to be analyzed The probe nucleic acid is separated and removed; and (6) detecting the hybridization message on the substrate via step (5). The rapid nucleic acid fragmentation analysis described in claim 44 of the patent application, wherein the matrix is a film. A method for rapid nucleic acid fragmentation analysis according to claim 45 of the patent application, 36 200806799 wherein the film is a nylon membrane. 47. The rapid nucleic acid fragmentation analysis method according to claim 45, wherein the shaft in the step (1) is an H fiber paper. 48. A method of assay for rapid nucleic acid mussels as described in the patent specification, private, #, 牝 or s, wherein the matrix has a pore diameter of from G.1 to 50 μη/. Milk, such as the application of _ 4 彳之快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快快5. The method for rapid nucleic acid fragmentation analysis described in Patent Application No. 44, and the nucleic acid to be analyzed in the eighth step (2) is ribonucleic acid. Shenyue specializes in iliiil 44; 快速 described rapid nucleic acid crush analysis method, eight steps (3) is to place the substrate at 8 ° ° C to 13 (TCT, drying! for 10 minutes. 52, such as Shen ^ The fast core described in item 44 of the patent (4), = medium = the substrate is irradiated with ultraviolet light for 5 to 1 〇, and the substrate is further dried. 53. As shown in the patent application, the rapid nucleic acid printing of step (4) The collapse analysis method, 54) the Qianji pairing reaction time is 2 to 5 minutes. For example, in the patent application range (3), the buffer solution is a nucleic acid printing and analyzing method. The sodium sulphate solution, the sputum 〇〇 5 to 0.15 times the standard 榕榕 55, as claimed in the patent range, the fast description Nucleic acid imprinting analysis method, 200806799 wherein the buffer solution in step (5) further comprises 〇〇5% to 〇15% (w/v) sodium dodecyl sulfate solution. 56, as in claim 44 The rapid nucleic acid fragmentation analysis method, wherein the rinsing time of the buffer solution in the step (5) is 3 to 6 minutes. 57. A rapid nucleic acid printing analysis method comprising the following steps: (1) providing a substrate The matrix has pores; (2) delivering a probe nucleic acid to the substrate to adsorb the probe nucleic acid to the substrate; (3) immobilizing the probe nucleic acid on the substrate, and The substrate is in a dry state; (4) a solution containing the nucleic acid to be analyzed is interacted with the substrate of step (3) at 4 ° C to 70 ° C to cause the probe nucleic acid to be treated on the substrate Analyze the nucleic acid for base pairing for a few minutes; (5) the substrate of step (4) , rinsing with a buffer solution to separate and remove the nucleic acid to be analyzed that is not bound to the probe nucleic acid; and (6) detecting a hybridization message on the substrate according to the step (5). The rapid nucleic acid fragmentation analysis method according to the item 57, wherein the substrate is a film. 59. The rapid nucleic acid fragmentation analysis method according to claim 58, wherein the film is a nylon film. The patent application of the 58th item of the rapid nucleic acid printing / shellfish method, 38 200806799 wherein the film in the step (1) - nitrocellulose paper. 61 62 63 64 65 66 67 68 士申明 patent scope 57, 58 The rapid nucleic acid fragmentation analysis method described in the item 59 or 6 (), wherein the pore of the substrate is 0.1 to 50 Å. The rapid nucleic acid fragmentation analysis method described in claim 57, wherein the step (3) The substrate is placed under the voyage for a period of 1 to 10 minutes. The rapid nucleic acid fragmentation analysis method described in claim 57, wherein the step (3) is to irradiate the substrate with ultraviolet light. After illuminating for $ to 1 minute Further, the substrate is dried. The rapid nucleic acid fragmentation analysis method according to claim 57, wherein the base pairing reaction time of the step (4) is 2 to 5 minutes. The rapid nucleic acid fragmentation analysis method according to Item 57, wherein the buffer solution in the step (5) comprises a 〇〇5 to 〇15 times standard sodium citrate solution, as described in claim 65 of the patent scope. The nucleic acid blotting analysis method, wherein the buffer solution in the step (5) further comprises a 0.05% to 〇15% (w/v) sodium dodecyl sulfate solution. The rapid nucleic acid fragmentation analysis method according to claim 57, wherein the buffer solution has a rinse time of 3 to 6 minutes in the step (5). A rapid nucleic acid fragmentation analysis method comprising the steps of: (1) providing a matrix having pores; 39 200806799 (2) transferring a nucleic acid to be analyzed to the substrate, and causing the nucleic acid to be analyzed to be (3) immobilizing the nucleic acid to be analyzed on the substrate; (4) adding a solution containing the probe nucleic acid to the substrate in a pressure difference manner; and (3) / on the substrate to make the probe The nucleic acid rapidly enters the matrix and is paired with the nucleic acid to be analyzed on the substrate for several minutes. (5) separating and removing the probe nucleic acid in step (4) that is not bound to the nucleic acid to be analyzed; And (6) detecting the hybridization message on the substrate via step (5). 69 70 71 72 73. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the probe nucleic acid in the step (4) is vacuumed on the other surface of the substrate, and formed by vacuum Negative pressure causes the probe nucleic acid to enter the interior of the matrix. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the probe nucleic acid in the step (4) is in a flow channel to which the substrate is attached, and enters the inside of the matrix in a pressurized manner. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the substrate in the step (1) is dry. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the substrate in the step (1) is a film. The rapid nucleic acid fragmentation analysis method according to claim 72, wherein the substrate is a nylon membrane. /, 40 200806799 74. The rapid nucleic acid fragmentation analysis method according to claim 72, wherein the matrix is mononitrocellulose paper. 75. A method of rapid nucleic acid flash analysis as described in claim 68, 72, 73 or 74, wherein the matrix has a pore diameter of from 1 to 50 μm. 76. The method of claim 1, wherein the nucleic acid to be analyzed in the step (2) is a deoxyribonucleic acid. 77. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the nucleic acid to be analyzed in the step (2) is a ribonucleic acid. 78. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the step (3) is performed by placing the substrate at a temperature of 80 ° C to 130 ° C to perform the analysis of the nucleic acid to be analyzed. Fixed to the substrate. 79. The rapid nucleic acid fragmentation analysis method according to claim 78, wherein the drying time of the step (3) is 1 to 1 minute. 80. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the step (3) irradiates the substrate with ultraviolet light to fix the nucleic acid to be analyzed on the substrate. 81. The rapid nucleic acid fragmentation analysis method according to claim 10, wherein the substrate to which the nucleic acid to be analyzed is immobilized is further dried after the step (3). 82. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the base pairing reaction time of the step (4) is 2 to 5 minutes. 41 200806799 83 84 85 86. The rapid nucleic acid fragmentation analysis method according to claim 68, wherein the step (5) is performed by rinsing with a low ionic strength buffer solution. The rapid nucleic acid fragmentation analysis method according to claim 83, wherein the low ionic strength buffer solution in the step (5) comprises a standard sodium citrate solution of 5 to 0.15 times. The method according to claim 84, wherein the low ionic strength buffer solution further comprises 〇〇5% to 0.15% (w/v) of dodecyl sulfate in the step (5). Sodium solution. -, ·, as claimed in claim 83, wherein the low ionic strength buffer solution has a rinsing time of 3 to 6 42 in the step (5).