200934876 九、發明說明： 【發明所屬之技術領域】 本發明與限制㈣酶作輕度的測定或檢驗方法有關， 特別是指-種低成本、快速、高準確性之定量印痕基因基因 曱基化方法者。 【先前技術】 〇 隨著醫學分子生物學的發展，研究者已經發現許多遺傳 疾病的發生其實都與細胞内基因的調控異常有關，基因的缺 失或疋外基因的修飾（epigenetic modification)都會影響基因的 •正常表現，因此開發各種基因檢測技術將有助於分子醫學的 深入研究，同時也將提供人類遺傳疾病篩檢及分子診斷所需 工具； 〇 在已知會影響人類基因表現的各種因素中，外基因的修 飾是較少人深入研究的一種現象，這類的修飾不像基因突變那 樣會改變DNA序列，而是透過對基因體的外加調整來控制基因的 開與關，因此是細胞中決定何種基因需要表現，何種基因應該休 息的調節機制之一。目前已知的外基因修飾現象包括DNA的甲 基化(methylation)、染色質的構造(chromatin structure)及 histone 蛋 白貝的乙酿化（acetylation)等等； 基因印痕(genetic imprinting)是一種較為大家所熟知的外基 200934876 因修,飾現象，其利用基因的甲基化來調節基因的表現，而使用這 種调印表現方式的基因就稱為印痕基因，其特點是依據基因來自 〆、系或疋母系而决疋對偶基因⑽如）的表現與否。然而當印 痕基口中4被Ψ基化的對偶基因甲基化不完全，或是不該被 甲基化的對偶基因被部份或完全甲基化時，都會造成基因的 不正¥表現’許多罕見遺傳疾病的發生便是由於印痕基因的 異常甲基化所導致； 以印痕基因座（Z/77或稱尤CA^/〇77)為例，其位在人類 U號染色體15· 5位置上’正常情況下，基因座中來自母親 的對偶基B)會在上游位置被完全甲基化，而來自父親的對偶 基因在上游相對位置則不會被甲基化，因此一般正常人在此 基因座上被甲基化的程度約在50%左右。然而當來自母親 的對偶基因因為某些因素而導致甲基化不完全時，會造成印 & 痕基固M (UTJ/KCNQJOTJ)的低复ψ基化 (hyp〇methylati〇n)，這樣的現象已知是造成罕見遺傳疾病貝 克威斯早德曼氏症（Beckwith-Wiedemann Syndrome ; BWS )最 主要的原因，因此如果能夠發展合適的分子檢測技術檢測印 痕基因座（Z /77/尺CA^7 077 )的曱基化程度，便能夠對貝克威 斯韋德曼氏症進行診斷，甚至於產前篩檢； 針對去氧核糖核酸擷取印痕基因並觀察的技術，過去多 仰賴南方墨點法（Southern blot )，此技術是先用限制酶將去 7 200934876 氧核糖核酸切割成片段，㈣體電泳將各片段分離後，把片 段轉移至濾』’料熟錢射線同位素標定的探針進行雜 合反應後’以自動顯影技術來_所要檢驗的片段，藉此對 特定印痕基因的甲基化程度進行檢測； 不過南方墨點法具有幾項缺點，首先，此方法所需使用 的去氧核糖核酸（職）量相當大，約需ig〜i5微克㈤200934876 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the limitation (4) that the enzyme is used for a light measurement or test method, in particular, a low-cost, fast, high-accuracy quantitative imprinting gene gene thiolation Method. [Prior Art] With the development of medical molecular biology, researchers have discovered that the occurrence of many genetic diseases is actually related to the abnormal regulation of intracellular genes, and the deletion of genes or epigenetic modification affects genes. • Normal performance, so the development of various genetic testing techniques will facilitate the in-depth study of molecular medicine, and will also provide the tools needed for screening and molecular diagnosis of human genetic diseases; 〇 among the various factors known to affect human gene expression, The modification of the foreign gene is a phenomenon that is less studied by more people. Such a modification does not change the DNA sequence like a genetic mutation, but controls the opening and closing of the gene by external adjustment of the genome, so it is determined in the cell. One of the regulatory mechanisms for which genes need to be expressed and which genes should rest. The currently known external genetic modification phenomena include methylation of DNA, chromatin structure, and acetylation of hisine protein; genetic imprinting is a relatively common phenomenon. The well-known external base 200934876 uses the methylation of genes to regulate the expression of genes. The gene using this type of imprinting is called the imprinted gene, which is characterized by the gene from the sputum and the system. Or the maternal line and the performance of the dual gene (10) such as). However, when the methylation of the thiolated dual gene in the imprinted base is incomplete, or the dual gene that should not be methylated is partially or completely methylated, it will cause the gene's misbehaving performance. The occurrence of genetic diseases is caused by the abnormal methylation of the imprinted gene; taking the imprinted locus (Z/77 or especially CA^/〇77) as an example, it is located at the human U chromosome 15·5 position' Normally, the dual base B from the mother in the locus will be completely methylated in the upstream position, while the dual gene from the father will not be methylated in the upstream relative position, so the normal human is at this locus. The degree of methylation is about 50%. However, when the maternal gene from the mother is incompletely methylated due to certain factors, it will cause low retanning (hyp〇methylati〇n) of the imprinted M (UTJ/KCNQJOTJ). The phenomenon is known to be the most important cause of the rare genetic disease Beckwith-Wiedemann Syndrome (BWS), so if you can develop appropriate molecular detection techniques to detect the imprinted locus (Z / 77 / 尺 CA ^ The degree of thiolation of 7 077 ) enables the diagnosis of Beckwith Wedmann's disease, even prenatal screening. The technique of extracting and scanning the DNA for DNA has relied on southern ink dots. Southern blot, which uses a restriction enzyme to cleave the oligodeoxyribonucleic acid into a fragment, and (4) electrophoresis separates the fragments and transfers the fragments to a probe that is calibrated by the spent ray isotope calibration. After the hybrid reaction, the fragment to be tested by the automatic development technique is used to detect the degree of methylation of the specific imprinted gene; however, the southern dot method has several disadvantages. First, this method Deoxyribonucleic acid to be used (post) is rather large, about ig~i5 micrograms (v)
G 左右’對於只能萃取少量去氧核糖核酸（職）的樣本，如 胎兒的絨毛或羊水細胞並不適用；其次，南方墨點法步驟繁 雜’操作上既費時X耗力，進行時間該天至—個禮拜才可 完成；最後’實驗中所制的放射線同位素具有危險性，為 一種致癌性物質’且單次檢測的花費成本約在新台幣兩千元 左右，既危險成本又高。 因此’發展-套簡便、快速、安全、低成本又只需少量 去氧核糖核酸（DNA)樣本的甲基化定量檢測方法，不論對 ,Ο料上的基礎研究或是臨床上的料應用都有其存在的價 . 值。 【發明内容】 本發明目的在提供—種簡便、快速、安全的甲基化定量 檢測方法，利用曱基化敏感性限制内切酶(methylati〇n論e restriction enclo皿dease)先對去氧核糖核酸（DNA)進行處理後， 8 200934876 以定量聚合_鎖反應比較印痕基因座上兩段分別具有及 T具有甲基錄祕限___触之去氧核糖核酸 職）片段的臨界循環數差值，由兩者差異即可計算出美 因座上去氧核糖核酸⑽A)甲基化的比例，判斷甲基：_ 度； 重要的是，此方法可在由戟羊水或絨域細胞中所取 得的少量醜中分析其印痕基因上的甲基化程度，因此本 方法可同時在產前及產後的樣本中操作，具有相當高的臨床 應用價值，為省時省力、成本低廉，又符合經濟效益之方法。 【實施方式】 本發明快速定量印痕基因曱基化的方法，其針對印痕基 因座U/77/i：CW27077)的實施步驟，請配合第丄圖所示，依 序如下： 〇 限制内切酶處理（1 0 ): 將自樣本中所萃取出之去氧核糖核酸（DNA)以曱基化敏 感性限制内切酶你(1 0 0 )處理，待其作用完全後，再利 用升溫去除該曱基化敏感性限制内切酶(AbilX 1 〇 〇 )之活性。 其中’該較佳的實施例’係將約0.5-1微克（yg)的去 氧核糖核酸（DNA)，與1 0活性單位（U)的曱基化敏感性限制 内切酶(AWI) ( 1 〇 〇 )、3微升(// 1 )的十倍(10 X)曱基化敏感性 200934876 限制内切酶(y〇iI) ( 1 〇 〇 )緩衝液及不含去氧核糖核酸酶 (DNAase-free)之二次水混合’使溶液最後之體積為3 〇微升（# 1 )，置於37°C的環境下，反應1-1.5小時後，整體溫度提 升至65°C下20-45分鐘’以去除甲基化敏感性限制内切酶(姚^i) (10 0)之活性； 定量聚合酶鏈鎖反應（Quantitative polymerase ehain reaction = qPCR) ( 2 0 ): O 利用定量聚合酶鏈鎖反應以曱基化敏感性限制内切酶(撕^ι) (1 0 0 )處理過之去氧核糖核酸（DNA)為模板，針對印痕基 因座（Ζ/77ΑΚΓ7νρ/〇77)之5，端兩段分別具有及不具有單一 ' 個曱基化敏感性限制内切酶( 1 〇 〇 )辨識切位之片段進行 擴增反應； 引子對（primers)設定（2 1 ):進行定量聚合酶鏈鎖反 應時，所需使用之引子對為約20個鹼基的寡核苷酸辦，其 ❹ 5’到3’端之序列分別為： (1)含有甲基化敏感性限制内切酶(#况1) ( 1 〇 〇 )辨識切 位 之片段 的正向 引 子，為 5’ -AACATCCCGATCCCCTGCAT-3，，反向引子為 5’ -GGCCAGTTCTCTGCGTGATG-3’ ； (2)不含有曱基化敏感性限制内切酶(他①（1 〇 〇 )辨識 切位之片段的正向 引子為 200934876 5’ -AATGGGGATGTGAGGATCAGG-3’，反向引子為 5’ -TGACCCCAGTGGAATATGTGC-3，。 反應進行（2 2 ):將各引子對分別與甲基化敏感性限制 内切酶(TVb/I) ( 1 〇 〇 )處理後之去氧核糖核酸（dna)、榮光標 定物質、去氧核糖核酸（DNA)聚合酶以及反應所需緩衝液進行混 合後，將此二溶液置入溫度循環器進行反應，請配合第2圖 所示； Ο 啟始狀態（2 2 0 ):溫度首先在94-95°C保持10-20 分鐘； 三段式循環（2 2 1 ):再進行50次三段式反應的循 環，即94-95°C，保持5-15秒，目的係在使去氧核糖核酸 (DNA)雙股分離，降溫至55-60°c保持15—25秒，目的係在 使各引子對與其相對應之印痕基因序列進行配對枯合，再升 至70-72 C保持15-30秒，其目的係在使去氧核糖核酸 .❹ （DNA)聚合酶進行反應予以DNA複製； * 終止狀態（2 2 2 ):最後又在94-95°C保持10-20秒； 其中，較佳的三段式反應實施例是將溫度在94〇c保持 1〇秒，雙股去氧核糖核酸（DNA)分離，其後改變到58〇c保 持20秒，各引子對與相對之印痕基因序列進行配對粘合， 形成去氧核糖核酸（DNA)聚合酶辨識並起始作用的位置，其 中包括兩個擴增片段：不含有曱基化敏感性限制内切酶(他圯（工 11 200934876 0 0 )辨識切位之片段以及含有曱基化敏感性限制内切酶(愚出 (1〇 0)辨識切位之片段，即為不含酶辨識切位片段（丄〇丄） • 以及含酶辨識切位片段（1 0 2 )，請配合第3圖所示，最後再 改變到72 C保持30秒’使去氧核糖核酸（DNA)聚合酶進行反 應，達成去氧核糖核酸（DNA)片段複製的目的，每次三段式 反應就能使基因片段的數量成雙倍，而在不斷雙倍複製的過 程中，嵌入去氧核糖核酸（DNA)的螢光劑之數量也隨之增 ❹ 加。 如果預設一個螢光量，而將此兩溶液反應進行達到此螢 光1時所需的循環數稱為臨界循環數（Ct;Cyde threshold)，可 A L界循％數將與溶㈣原有料㈣糖彳嫌⑽A)模板 數里呈反等比關係，意即反應所需的臨界循環數每多一個， 就表示其難去氧核糖減(職)的數量餘對少一半。 由於不含酶辨識切位片段（1 0 1 )並不會被甲基化敏感性 -限制内切酶陶）（1 〇 〇 )所切割，因此其所有去氧核糖核酸 ' )片段都可充當定量聚合酶鏈鎖反應時的去氧核糖核 (NA)拉板’即所有的去氧核糖核酸⑽a)模板數量相當 ;甲基化及未甲基化去氧核糖核酸⑽A)的總和； 相對的，含酶辨識切位諸（1 〇 2)如果未被甲基化， m限制内切喊理（10)時會被甲基化敏感性限制内切酶 1 0 0)所辨識切割，無法再充當定量聚合酶鍵鎖反 12 200934876G around 'for samples that can only extract a small amount of DNA (job), such as fetal villi or amniocytes are not suitable; secondly, the southern ink point method is complicated 'operation time is time-consuming X effort, the time of the day It can only be completed in a week. Finally, the radioisotope produced in the experiment is dangerous and is a carcinogenic substance. The cost of a single test is about NT$2,000, which is dangerous and high. Therefore, 'development - a simple, fast, safe, low-cost method for the quantitative detection of methylation of small amounts of DNA samples, whether basic, basic research or clinical application There is a price of its existence. SUMMARY OF THE INVENTION The object of the present invention is to provide a simple, rapid and safe method for quantitative detection of methylation, using a thiolation sensitivity restriction endonuclease (methylati〇n on e restriction enclo dish) first on deoxyribose After treatment of nucleic acid (DNA), 8 200934876 compares the critical cycle number difference between the two segments of the imprinted locus and the T-DNA fragment of the T-reported DNA fragment. The value, the difference between the two can be calculated to determine the methylation ratio of DNA (10) A) on the stem of the US, to determine the methyl: _ degree; importantly, this method can be obtained from amniotic fluid or velvet cells A small amount of ugly analysis of the degree of methylation on the imprinted gene, so the method can be operated simultaneously in prenatal and postnatal samples, has a relatively high clinical application value, saves time and effort, low cost, and is economical The method. [Embodiment] The method for rapidly quantifying the thiolation of the imprinted gene of the present invention, and the implementation procedure for the imprinting locus U/77/i: CW27077), as shown in the figure, is as follows: 〇 restriction endonuclease Treatment (10): Treat the DNA (DNA) extracted from the sample with the thiolation sensitivity restriction endonuclease (10). After the effect is complete, remove the temperature by using the temperature. The thiolation sensitivity limits the activity of the endonuclease (AbilX 1 〇〇). Wherein the 'preferential embodiment' is about 0.5-1 microgram (yg) of deoxyribonucleic acid (DNA), and 10 activation units (U) of thiolation sensitivity restriction endonuclease (AWI) ( 1 〇〇), 3 microliters (// 1 ) ten times (10 X) thiolation sensitivity 200934876 restriction endonuclease (y〇iI) ( 1 〇〇) buffer and no DNase (DNAase-free) secondary water mixing 'The final volume of the solution is 3 〇 microliters (# 1 ), placed in an environment of 37 ° C, after 1-1.5 hours of reaction, the overall temperature is raised to 65 ° C 20-45 minutes' removal of methylation sensitivity restriction endonuclease (Yao ^i) (10 0) activity; quantitative polymerase chain reaction (Quantitative polymerase ehain reaction = qPCR) (20): O utilization quantification The polymerase chain reaction is based on the thiolation sensitivity restriction endonuclease (1 0 0 ) treated deoxyribonucleic acid (DNA) as a template for the imprinted locus (Ζ/77ΑΚΓ7νρ/〇77) 5, the end of the two segments with and without a single 'thiolated sensitivity restriction endonuclease (1 〇〇) identification of the cleavage segment for amplification reaction; primer pairs (primers) set (2 1) : When performing quantitative polymerase chain reaction, the primer pair used is about 20 base oligonucleotides, and the sequence from the 5' to the 3' end is: (1) sensitive to methylation Sex restriction endonuclease (# condition 1) (1 〇〇) recognizes the forward primer of the cleavage fragment as 5'-AACATCCCGATCCCCTGCAT-3, and the reverse primer is 5'-GGCCAGTTCTCTGCGTGATG-3'; (2) No The forward primer containing the thiolation sensitivity restriction endonuclease (the 1(1 〇〇) recognition cleavage fragment is 200934876 5' -AATGGGGATGTGAGGATCAGG-3', and the reverse primer is 5'-TGACCCCAGTGGAATATGTGC-3. Perform (2 2 ): deoxyribonucleic acid (dna), lens cursor, and deoxyribonucleic acid after treatment of each primer pair with methylation-sensitive restriction endonuclease (TVb/I) ( 1 〇〇) After the (DNA) polymerase and the buffer required for the reaction are mixed, the two solutions are placed in a temperature circulator for reaction, as shown in Fig. 2; 启 Start state (2 2 0 ): temperature first at 94- 95 ° C for 10-20 minutes; three-stage cycle (2 2 1 ): 50 more three-stage reactions Ring, 94-95 ° C, for 5-15 seconds, the purpose is to separate the DNA strands (DNA) double-strand, cooling to 55-60 ° c for 15-25 seconds, the purpose is to make each primer pair The corresponding imprinted gene sequence is paired and homogenized, and then raised to 70-72 C for 15-30 seconds, the purpose of which is to make DNA replication by deoxyribonucleic acid (DNA) polymerase; * termination state (2 2 2 ): Finally, at 94-95 ° C for 10-20 seconds; wherein the preferred three-stage reaction example is to maintain the temperature at 94 ° C for 1 〇 second, double stranded deoxyribonucleic acid ( DNA) is isolated, and then changed to 58〇c for 20 seconds. Each primer pair is paired with the opposite imprinted gene sequence to form a location where the DNA polymerase recognizes and initiates action, including two. Amplified fragment: does not contain a thiolation sensitivity restriction endonuclease (His 工 (工11 200934876 0 0) identifies the cleavage fragment and contains a thiolation sensitivity restriction endonuclease (folly (1〇0) Identify the fragment of the cleavage, ie, the cleavage fragment without the enzyme (丄〇丄) • and the cleavage fragment with the enzyme identification (1 0 2 ) Please match the picture shown in Figure 3, and finally change to 72 C for 30 seconds to allow the DNA polymerase to react to achieve the purpose of DNA fragment replication, each three-stage reaction. The number of gene fragments can be doubled, and the number of fluorescent agents embedded in deoxyribonucleic acid (DNA) increases during the double replication process. If a fluorescence amount is preset, and the number of cycles required to react the two solutions to reach the fluorescence 1 is called the critical cycle number (Ct; Cyde threshold), the AL boundary can be used to dissolve the (four) original material (4). The glycoside (10)A) is inversely proportional in the number of templates, meaning that each additional number of critical cycles required for the reaction means that the number of difficult DNA depletions is less than half. Since the enzyme-free cleavage fragment (1 0 1 ) is not cleaved by the methylation-sensitive endonuclease (1 〇〇), all of its DNA fragments can act as The number of templates for the deoxyribonucleic acid (NA) pull plate at the time of quantitative polymerase chain reaction is equivalent to that of all deoxyribonucleic acid (10) a); the sum of methylated and unmethylated deoxyribonucleic acid (10) A); If the enzyme-containing cleavage position (1 〇2) is not methylated, the m restriction endonuation (10) will be recognized by the methylation sensitivity restriction endonuclease 1 0 0), and can no longer be Acting as a quantitative polymerase keylock anti-12 200934876
應時的去氧核糖核酸⑽A)模板，而在含酶辨識切位片段G —中/、有甲基化的去氧核糖核酸（DNA)可以被當成去 氧核糖核酸（DNA)模板進行擴增。 數據計算（2 3 ): 由以含酶辨識切位片段（i 〇 2)與不含酶辨識切位片段（丄 〇 1 )為去氧核糖核酸（DNA)模板所得的臨界循環數差值，可 以計算出印痕基因座⑽歡聊^7)上甲基化的程度，即 該含酶辨識切位片段（i 〇 2 ).内己f基化時，該甲基化敏感性 限制内切酶(胸 1)( 1⑽）即無法對其進行辨識切割，而可繼續 成為去氧核糖核酸（DNA)之模板進行擴增；而若未甲基化時，則 含酶_她片段（i 〇 2 )内之片段即會以基化敏感性限制 内切酶_) ( i 〇 〇 )所辨識並切割，該未甲基化之含酶辨識切 位片段（1 0 2 )即無法成為去氧核糖核酸(DNA)之模板繼續進 仃擴增，因此在等量去氧核糖核酸⑽A)中，可能會因為含該含 酶辨識切位片段（1〇 2)之甲基化程度不同，而形成臨界循環 數值的變差異；而不含酶辨識切位片段（丄〇丄），因完全不受 甲基化敏雜限伽切酶的影響，因此可被完全的擴增，形成: 對知組。藉由擴增等量的含_辨識她片段（丨Q 2 )及不含酶 辨識切位片段（1 Q 1)使制達定量㈣光量時，將所需之臨 界循環數值進行比較’即可得知該含酶辨識切位片段（i 0 2) 曱基化的程度； 13 200934876 例如含酶辨識切位片段（1 ◦ 2 )的臨界循環數為10，而 不含酶辨識切位片段（1 0 1 )的臨界循環數為9，表示不含 酶辨識切位片段（i i)的擴增數目在成為29=512倍後榮光 量達到預設值，而含_識她㈣（i Q 2 )則必須要成為 2〗°=1〇24倍後螢光量才能達到預設值，表示已甲基化部分的 去氧核糖核酸⑽A)比例佔所有去氧核糖核酸⑽A)(有甲 基化+未甲基化）的一半，可知個體在此基因座上甲基化的程 度為1/21" = 50%，為預期正常範圍值； 相對的’若是含酶辨識切位片段（1 〇2)有低度甲基化之 現象時，該含酶辨識她片段（i ◦ 2 ) f基化可為擴增的去氧 核糖核酸⑽A)模板減少，故所界循環數必贿之增 加’因此其可能的臨界循環數為1卜衫含_識切位片段 ^ ο 1)所有的去氧核糖核酸（DNA)模板均可被擴增，因此 Q 八界循^數較小，或為9 ’表示含酶辨識切位片段（1 〇 2 ) 必，要成為2丨丨％2048倍後螢光量才能達到預設值，已甲基 化。卩刀的比例佔所有去氧核糖核酸（dna)量的 為不正$的曱基化範圍值，因此當含酶辨識切位片段（1 2 )的臨界循環數為A ’不含酶辨識切位片段（1 〇 1 )的臨 :衣數 =B時，已甲基化部份為全部*氧核糖核酸（DNA) 量的^倍，即曱基化比例為100 X 1/2Α—Β%。經過計算， 在正吊人’其印痕基因座⑽攸C聊077)上含酶辨識切位片 14 200934876 段（1 0 2 )與不含酶辨識切位片段（丄〇 i )的臨界循環數差 值大約在+1左右，表示基因甲基化程度約為5〇%上下，符合 預期。 而此數據§十算（2 3 )係可如第4及5圖所示，其中， 第4圖係為一健康個體之實驗數據圖，其中，到達一螢光量 時’其含酶辨識切位片段（1 〇 2 )的臨界循環數為25. 77， 而不含酶辨識切位片段（1 0 1 )則為24.87，臨界循環數差值 為〇_ 9，甲基化之指數比例為53. 59% ;而如第5圖所示，其 係為印痕基因座(Z/77/^CA^/〇77)低度甲基化的病患取得 者其中，含酶辨識切位片段（1 0 2 )臨界循環數為28. 78， 而不s酶辨識切位片段（工〇工）之臨界循環數為25·32，而 兩臨界循環數之差值為3.46,因此曱基化之指數比例為 9. 09% 〇 、判斷（3 ◦).由該曱基化之比例，在5G%左右之數值，可視 ^一正常個體’而當比例明顯偏離該—數值，即為曱基化異常， -、中明顯小於该-數值時，為該基因座低度甲基化之表現。 知由於此種方法會使用聚合酶鏈鎖反應擴增去氧核糖核 ΝΑ)數量，所以只須少量去氧核糖核酸（dna)，即能夠 不=複製而達咖科光量’絲蚊量聚合賴鎖反應所 娜得的臨界循環數已f基化料佔全部去氧核糖核酸 15 200934876 =::==== …體操作之時間，除了前階段的去氧核糖核酸（舰)取 传幸乂費時外’其他時間均係計秒方式為之，數十循環之時間亦短， 故在-天之内即可完成，所耗_十分的短，符合快速偵測或是 快速診斷的需求； 成本極低，即整體之成本以耗材而論，約在新台幣一百元以 下而在人力以及時間之成本上，遠較傳統之方式節省五至七倍 的時間’故成本亦在傳統的四分之—至七分之—的低成本狀態下 者。 安全性高，本方法單純以螢光劑做為偵測之標的，過程 不使用放射性物質或其他有毒致癌物質，為一高安全性的方 法者； 另外’此種技術不只能用在印痕基因座（Ζ/77/^ΤΛ^/(977) 上，舉凡是會受曱基化修飾之基因皆能夠使用，另外，臨床 上此方法則可應用於判斷各種與曱基化異常可能有關的疾 病’如某些癌症。 200934876 【圖式簡單說明】 第1圖係為本發明之主要流程示意圖。 第2圖係為本發明之反應進行流程示意圖。 第3圖係為本發明之曱基化敏感性限制内切酶(Α/οίΙ)進行辨識切位 片段之示意圖。 第4圖係為本發明在印痕基因座（z/r7/s：CA^/〇77)正常甲基化 的臨界循環數示意圖。 〇 第5圖係為本發明在印痕基因座低度曱基化 的臨界循環數示意圖。 【主要元件符號說明】 《本發明》 限制内切酶處理（1〇) 甲基化敏感性限制内切酶(TVort) ( 1 0 0 ) 〇 不含酶辨識切位片段（1 0 1 ) 含酶辨識切位片段（1〇 2) 定量聚合酶鏈鎖反應（Quantitative polymerase chain reaction = qPCR) ( 2 〇 ) 引子對(primers)設定（2 1 ) 反應進行（2 2) 啟始狀態（2 2 0) 17 200934876 三段式循環（2 21) 終止狀態（2 2 2) 數據計算（2 3) 判斷（3 0 )Time-dependent DNA (10) A) template, and in the enzyme-containing cleavage fragment G - /, methylated DNA (DNA) can be amplified as a deoxyribonucleic acid (DNA) template . Data calculation (2 3 ): The difference in critical cycle number obtained by identifying the cleavage fragment (i 〇 2) with an enzyme and identifying the cleavage fragment (丄〇1 ) without a enzyme as a DNA template. It is possible to calculate the degree of methylation on the imprinted locus (10), ie, the enzyme-recognizing cleavage fragment (i 〇2). The methylation-sensitive restriction endonuclease (thoracic 1) (1(10)) cannot be identified and cut, but can continue to be a template for deoxyribonucleic acid (DNA) for amplification; if not methylated, it contains enzymes (her 片段2) The fragment is recognized and cleaved by the cleavage-sensitive restriction endonuclease _) (i 〇〇), and the unmethylated enzyme-containing cleavage fragment (1 0 2 ) cannot be deoxyribose The template of the nucleic acid (DNA) continues to expand, so in the same amount of deoxyribonucleic acid (10) A), the methylation degree of the cleavage fragment (1〇2) containing the enzyme may be different to form a criticality. The difference in the value of the cycle; without the enzyme to identify the cleavage fragment (丄〇丄), because it is completely unaffected by the methylation-sensitive gamma Thus amplified can be fully formed: of known groups. By amplifying an equal amount of _ identifying her fragment (丨Q 2 ) and identifying the cleavage fragment (1 Q 1) without enzymes, when the quantitative (four) light quantity is obtained, the required critical cycle values are compared. It is known that the enzyme-containing cleavage fragment (i 0 2) is thiolated; 13 200934876 For example, the critical cycle number of the cleavage fragment (1 ◦ 2) containing the enzyme is 10, and the cleavage fragment is not included in the enzyme identification ( The number of critical cycles of 1 0 1 ) is 9, indicating that the number of amplifications without the enzyme-recognized cleavage fragment (ii) reaches the preset value after becoming 29=512 times, and contains _ her (4) (i Q 2 ) must be 2〗 ° = 24 times after the amount of fluorescence can reach the preset value, indicating that the methylated part of the DNA (10) A) proportion of all DNA (10) A) (with methylation + Half of the unmethylated), the degree of methylation of the individual at this locus is 1/21 " = 50%, which is the expected normal range value; the relative 'if the enzyme contains the cleavage fragment (1 〇 2) When there is a phenomenon of low methylation, the enzyme-containing recognition of her fragment (i ◦ 2 ) f-based can be reduced by the amplified DNA (10) A) template Therefore, the number of cycles is bound to increase the number of bribes. Therefore, the number of possible critical cycles is 1 衫 含 _ _ _ _ _ _ 1) All DNA samples can be amplified, so Q VIII The boundary number is small, or 9 ' indicates that the enzyme-containing cleavage fragment (1 〇 2 ) is necessary. To become 2丨丨%2048 times, the amount of fluorescence can reach the preset value and is methylated. The proportion of the sickle accounted for the value of the thiolation range of the amount of all the deoxyribonucleic acid (dna), so when the enzyme contains the cleavage fragment (1 2 ), the critical number of cycles is A 'the enzyme-free cut position. The fraction of the fragment (1 〇1): when the number of clothes = B, the methylated portion is twice the total amount of *oxyribonucleic acid (DNA), that is, the ratio of thiolation is 100 X 1/2 Α - Β %. After calculation, the critical cycle number of the enzyme-recognition cut-off piece 14 200934876 (1 0 2 ) and the enzyme-free cleavage fragment (丄〇i ) was included in Zhengshangren's imprinting locus (10)攸C. The difference is about +1, which means that the degree of gene methylation is about 5%, which is in line with expectations. The data § ten (2 3 ) can be as shown in Figures 4 and 5, wherein Figure 4 is an experimental data plot of a healthy individual, where the enzyme-recognition cleavage is reached when a fluorescent amount is reached. The critical cycle number of the fragment (1 〇 2 ) is 25.77, while the cleavage fragment without enzyme identification (1 0 1 ) is 24.87, the difference of the critical cycle number is 〇 _ 9, and the index ratio of methylation is 53. 59%; and as shown in Fig. 5, it is a patient with low methylation of the imprinted locus (Z/77/^CA^/〇77), which contains the enzyme recognition cleavage fragment (1) 0 2 ) The number of critical cycles is 28.78, and the number of critical cycles of the cleavage segment (worker) is 25.32, and the difference between the two critical cycles is 3.46, so the index of thiolation The ratio is 9.09% 〇, judgment (3 ◦). The ratio of the 曱 ,, in the value of about 5G%, can be regarded as a normal individual' and when the ratio deviates significantly from the value, it is a thiolated anomaly. , -, where the value is significantly less than the - value, is the performance of the low methylation of the locus. Knowing that this method will use the polymerase chain reaction to amplify the number of deoxyribonucleosides, so only a small amount of DNA is needed, that is, it can not be copied and the amount of light is reduced. The critical cycle number of the lock reaction has been f-based material accounted for all the deoxyribonucleic acid 15 200934876 =::==== ... the time of the body operation, in addition to the pre-stage DNA (ship) It takes time to count the other time, and the time of dozens of cycles is also short, so it can be completed within -day, the consumption is very short, which meets the requirements of fast detection or rapid diagnosis; Very low, that is, the overall cost is in terms of consumables, about NT$100 or less, and the cost of manpower and time is much lower than the traditional way to save five to seven times. The cost is also in the traditional four points. The low-cost state of - to seven-points. High safety, this method is purely using fluorescent agent as the target of detection. The process does not use radioactive substances or other toxic carcinogens, which is a high safety method. In addition, this technology can not only be used in the imprinting locus. (Ζ/77/^ΤΛ^/(977), all genes that can be modified by thiolation can be used. In addition, clinically, this method can be applied to judge various diseases that may be related to thiolation abnormalities. For example, some cancers. 200934876 [Simplified illustration of the drawings] Fig. 1 is a schematic diagram of the main flow of the present invention. Fig. 2 is a schematic flow chart showing the reaction of the present invention. Fig. 3 is a thiolation sensitivity of the present invention. Schematic diagram of restriction endonuclease (Α/οίΙ) for identifying cleavage fragments. Figure 4 is a schematic diagram showing the critical number of cycles of normal methylation at the imprinted locus (z/r7/s: CA^/〇77). 〇 Figure 5 is a schematic diagram of the critical cycle number of the low thiolation of the imprinted locus in the present invention. [Main element symbol description] "The present invention" restriction endonuclease treatment (1〇) within the methylation sensitivity limit Digestive enzyme (TVort) ( 1 0 0 ) 〇 Enzyme-recognized cleavage fragment (1 0 1 ) Enzyme-recognized cleavage fragment (1〇2) Quantitative polymerase chain reaction = qPCR ( 2 〇) Primer set (2 1 ) Reaction progress (2 2) Start state (2 2 0) 17 200934876 Three-stage cycle (2 21) Termination state (2 2 2) Data calculation (2 3) Judgment (3 0 )
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