TWI323285B - Identification of mei and plum processing product by pcr - Google Patents
Identification of mei and plum processing product by pcr Download PDFInfo
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1323285 九、發明說明： 【發明所屬之技術領域】 本發明係一種檢驗梅及李攙假之方法，以梅及 李核酸（DNA)上之差異設計引子（primer)，以聚合 酶鏈鎖反應（polymerase Chain reacti〇n，pCR)擴增 梅與李具差異之核酸片段，藉以達到檢測之目的。曰 【先前技術】 梅及李疋中國原生之果樹，過去對梅及李之研 究多著重於其加工上之應用，對其品種之分類方法 亦多是以其型態上之分類，以下將先前對梅、李區 分之研究及技術作一概述： 一、過去常用分類方法 過去用以分辨不同植物之方法主要是由外在之 型態等等去區分，如劉棠瑞1981年於國立編譯館 出版的植物分類學中第丨至86頁中提出傳統植 物为類法有型態分類法（Morphological taxonomic method)、解剖學分類法（Anatomical taxonomic method)、細胞學或細胞遺傳學分類法（Cyt0丨ogical taxonomic method)、化學分類法（Chemical 1323285 taxonomic method)、孢類學分類法（paiyn〇i〇gicai taxonomic method)、胚胎學分類法（Embryological taxonomic method)等六種分類法，而近年來生物技 術發展迅速，因此許多學者發展出分子標誌方法， 利用核酸序列的特異性區分不同物種，而這些分子 & δ志的方法有限制性片段長度多態性（Restricti〇n Fragment Length Polymorphism)、逢機增殖多型性 DNA (Random Amplified Polymorphism DNA, RAPD)、增殖片段長度多型性（Amplified Fragment Length Polymorphism)、序列特性增殖區域多型性 (Sequence-Characterized Amplified Regions)、簡單序 列重複擴增多型性（simple seqUence repeat)、簡單 序列重複區間擴增多型性（inter_simpie sequence repeat)或同功異構（is〇zytne)。 二、已用於梅或李分類之方法 在傳統分類法方面，黃卓治於198〇年台大食 科所碩士論文中根據梅子果實大小、形狀、顏色及 來源而將梅子區分為大粒梅、小粒梅、尖頭梅、平 頂梅四類，歐錫坤1995年於台灣農家要覽第151 至162頁中將台灣不同品種李子之果實依其色 澤、大小、顏色及糖酸度作區分。分子標誌方面， 方素嫣於2000年台大園藝所碩士論文中提到利用 1323285 _可有效地區分梅子種内十七種不 而利用不同的引子推奸p 種進 千進仃PCR後，觀察出現小 之片段而判斷品籍的猫—、夕 京林業大學學報第2〇卷】2至 年於北 的野生種及其近緣種桃纟纟j頁中㈣對梅 x 迎緣種桃李、杏、櫻共17個樣品的 系統發育進行RAPD研究，έ士杲甚§ -本 、 u呵九結果顯不李、性和梅的 系統發育過程中處於基本同等地位；而桃、櫻與梅 的親緣關係則隔較遠，汪祖華等人1991於園藝與 報第18卷第97至1G1頁中提到針對李、杏了: 之間的親緣關係及分類地位以同功酶作研究，認為 李、杏、梅應歸為同一屬或同一亞屬，而杏於系統 發育過程中介於李和梅之間，而且更親於梅。 常用分類序列1323285 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for testing plum and plum falsehood, designing a primer based on the difference between plum and plum nucleic acid (DNA), and polymerase chain reaction ( The polymerase Chain reacti〇n, pCR) amplifies the nucleic acid fragments of the difference between plum and plum for the purpose of detection.曰[Prior Art] Mei and Li Wei Chinese native fruit trees. In the past, the research on Mei and Li focused on the application of processing, and the classification methods of their varieties were mostly classified according to their types. An overview of the research and technology of the distinction between Mei and Li: 1. The past common classification methods used to distinguish different plants were mainly distinguished by external types, such as Liu Yurui published in the National Compilation Hall in 1981. Plant taxonomy, from page 丨 to page 86, suggests that traditional plants are Morphological taxonomic methods, Anatomical taxonomic methods, cytology or cytogenetic taxonomy (Cyt0丨ogical taxonomic) Method), chemical classification (Chemical 1323285 taxonomic method), paiyn〇i〇gicai taxonomic method, Embryological taxonomic method, and other six classification methods, and in recent years, biotechnology has developed rapidly Therefore, many scholars have developed molecular marker methods that use the specificity of nucleic acid sequences to distinguish different species, and these The method of sub- & δ zhi has Restricti〇n Fragment Length Polymorphism, Random Amplified Polymorphism DNA (RAPD), Amplified Fragment Length Polymorphism), Sequence-Characterized Amplified Regions, simple seqUence repeat, simple sequence repeat polymorphism (inter_simpie sequence repeat) or isoforms Structure (is〇zytne). Second, the method has been used for the classification of plum or plum In the traditional classification method, Huang Zhuozhi divided the plum into large grain plum and small grain plum according to the size, shape, color and source of plum fruit in the master's thesis of Taiwan University in 198. In the past, in 1995, in the Taiwanese farmer's book, pages 151 to 162, Ou Xikun distinguished the fruits of different varieties of plums in Taiwan according to their color, size, color and sugar acidity. In terms of molecular markers, Fang Suyi mentioned in the master's thesis of the NHK Institute of Horticulture in 2000 that the use of 1323285 can effectively distinguish 17 species of plums and use different primers to seduce p into thousands of sputum PCR, and observe the appearance of small The cat that judges the product by the fragment—the second volume of the Journal of Xijing Forestry University】The wild species of the north and the relative species of the peach aphid in the north of the 2nd year (four) on the plum x the edge of the peach, apricot, cherry A total of 17 samples were systematically developed for RAPD studies. The results of the έ - 本 本 本 本 本 本 本 本 本 本 本 本 u u u u u u u u u u u 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Far away, Wang Zuhua et al. in the horticulture and newspapers, Vol. 18, pp. 97 to 1G1, mentioned the relationship between Li and Apricot: the relationship between them and the taxonomic status were studied by the same enzymes, and they believed that Li, apricot and plum It should be classified into the same genus or the same subgenus, and the apricot is between Li and Mei during phylogeny, and is more intimate with Mei. Common classification sequence
Woese，C. R.等學者 1983 年在 Micr〇bi〇1 Rev.第47卷第621至669頁中曾提到，最有用 的为子標§志物質應同時存在於各物種，而Li，w. H. 專干者 1991 年於 Fundamental of molecular evolution中曾提到核糖體基因（rDNA)是真核生 物中可作為分類依據的物質之一，真核生物中真核 核糖體基因（rDNA)的構造如圖一，分別由小次單 位（small subunit, SSU)、大次單位（large subunit， LSU)及5S次單位（5Ssubunit)所組成，構造基因 1323285 之間為内轉錄間隔區（internal transcribed spacer， ITS)區域 ’ ITS 和基因間隔區（internally genetic spacer，IGS)的序列分析已利用在多種植物和微生物 上’如 Goel, S· S.等人 2002 年於 Molecular Evolution: Producing the Biochemical Data 中提到 以rDNA上的ITS序列比對，應用在菜豆與豇豆 的分類關係上，實驗結果成功的解決了在二個屬， 菜豆屬和紅豆屬之間種分配的問題，因為屬於菜豆 屬種的ITS序列都非常靠近，和豇豆屬是分離的， 而且菜豆和寬翼豆屬（Macroptilium)最相近，成功 的解決其複雜的傳統分類關係，以rDNA之its 序列作為分類者另有Wu, S. Y.等人2001年於J.Woese, CR et al., 1983, Micr〇bi〇1 Rev., Vol. 47, pp. 621-669, mentioned that the most useful sub-marker should be present in each species, and Li, w. H Specialists in the Fundamental of molecular evolution in 1991 mentioned that the ribosomal gene (rDNA) is one of the substances that can be classified in eukaryotes. The structure of eukaryotic ribosomal genes (rDNA) in eukaryotes is as follows. Figure 1, consisting of small subunit (SSU), large subunit (LSU) and 5S subunit (5Ssubunit), and the internal transcribed spacer (ITS) between the constructed genes 1323285 The sequence analysis of the region ' ITS and the ingenious genetic spacer (IGS) has been utilized in a variety of plants and microorganisms as described by Goel, S. S. et al., 2002, Molecular Evolution: Producing the Biochemical Data The ITS sequence alignment on rDNA was applied to the classification relationship between kidney bean and cowpea. The experimental results successfully solved the problem of species distribution between two genera, genus and red bean, because it belongs to the dish. The ITS sequences of the genus Beans are very close, separated from the genus Crotalaria, and the closest to the Macroptilium, successfully solving its complex traditional classification relationship, with the sequence of rDNA as the classifier. Wu, SY et al., 2001, J.
Hort. Sci. Biot.第76卷第127至132頁中所提 到的玫瑰屬、C〇x，A.V.等人1997年於PlantSyst.Hort. Sci. Biot. Vol. 76, pp. 127-132, of the genus Rose, C〇x, A.V. et al., 1997, PlantSyst.
Evol第208卷第197至223頁中所提到的拖鞋 蘭等。 【發明内容】 本發明目的及優點將部分描述於下，或可由描 述中顯而易見。本發明主要是針對梅及李之核糖體 核酉夂基因中内轉錄第一間隔區（簡稱汀S”為目標 片段進行試驗，對梅子品種及李子品種建立完整目 標核酸序列，比對其核酸序列’根據兩者序列之差 1323285 異性’設計引子對，藉以達到檢驗區分梅子及李子 加工品之目的。 、被式聚合酶鏈鎖反應（mUtipiex pcR) 使用如人已發表的通用引子（universal primer) 對16種梅及8種李rDNA上的ITS1區域進行 pcr，並將所得產物定序，發現在ITS 1片段上第 129 bp之位置上有連續丨8個核苷酸對的差異並 且在台灣栽培之16種梅品系與8種李品系上其差異 具一致性，因此在此區域設計一引子dITS1 (SEq 1〇 N01)，此引子僅可與李子之DNA模板進行配對， 因此對於梅與李具有區別性，搭配White et al.， (1990)所設計之引子 ITS1 (SEQ ID N02)及 ITS2 (SEQ ID N〇3)，使用引子之示意圖如圖二、圖三所 不，ITS 1與ITS2引子對經由聚合酶鏈鎖反應後，可 於梅與李分別得311 bp及329 bp核酸片段產 物，且由於進行複式聚合酶鏈鎖反應之故，其中僅 有李可由dITSI與ITS2弓丨子對得到一個約2〇〇 bp的核酸片段產物，因此於電泳時梅子僅可產生一 冗帶，而李子可產生兩亮帶，且片段大小差異高達 100 bp以上’可以直接判別出來。 一、利用即時定量聚合酶連鎖反應偵測系統 (LightCycler System)檢測 本發明為省去傳統聚合酶鏈鎖反應後需再經由 1323285 電泳分析等繁複耗時手續，又需兼顧鑑別檢測之精 確性，故尋求以即時定量聚合酶連鎖反應偵測系統 (LightCycler System)及赛巴格林（SYBR Green)螢 光染劑測定樣品之熔解溫度用以區別鑑定梅品系與 李品系。 首先以梅及李ITS區域上具差異之核酸片段Slippers, etc. mentioned in Evol Vol. 208, pp. 197-223. SUMMARY OF THE INVENTION The objects and advantages of the invention will be set forth in part or in the description. The invention mainly aims to test the first fragment of the inner transcribed region of the ribosome nuclear gene of plum and plum (referred to as T-S), and establish a complete target nucleic acid sequence for the plum cultivar and the plum cultivar, compared with the nucleic acid sequence thereof. 'Design the primer pair according to the difference between the two sequences 1323285', in order to achieve the purpose of testing and distinguishing the plum and plum processed products. The polymerase chain reaction (mUtipiex pcR) uses the universal primer published by humans. PCR was performed on the ITS1 region of 16 species of plum and 8 kinds of Li rDNA, and the obtained product was sequenced. It was found that there was a continuous 丨8 nucleotide pair difference at the 129 bp position on the ITS 1 fragment and cultivated in Taiwan. The 16 plum lines are consistent with the 8 varieties, so a primer dITS1 (SEq 1〇N01) is designed in this region. This primer can only be paired with the DNA template of plum, so it has The difference is matched with the primers ITS1 (SEQ ID N02) and ITS2 (SEQ ID N〇3) designed by White et al., (1990). The schematic diagrams using the primers are shown in Figure 2 and Figure 3. ITS 1 and ITS2 After the reaction by polymerase chain reaction, the 311 bp and 329 bp nucleic acid fragment products can be obtained from plum and plum respectively, and because of the double polymerase chain reaction, only Li can be obtained by dITSI and ITS2. A nucleic acid fragment product of about 2 bp, so that when the electrophoresis, the plum can only produce a redundant band, and the plum can produce two bright bands, and the difference in fragment size is as high as 100 bp or more 'can be directly discriminated. The polymerase chain reaction detection system (LightCycler System) detects the complicated time-consuming procedures such as electrophoresis analysis after the conventional polymerase chain reaction is omitted, and needs to take into account the accuracy of the identification detection. The melting temperature of the sample was determined by the polymerase chain reaction detection system (LightCycler System) and SYBR Green fluorescent dye to distinguish the plum and plum lines. First, the difference between the plum and the ITS region Fragment
没汁引子對 sITSl(SEQIDN04)及 SITS2(SEQID N05)’如圖四所示，圖五為以即時定量聚合酶連鎖 反應偵測系統（LightCycler System)及賽巴格林螢 光染劑求出以sITS1及sITS2引子對擴增梅與之 核酸片段產物之熔解溫度，由圖中可發現梅子與李 子之核酸片段產物之熔解溫度波峰皆相當地明顯， 梅的熔解溫度約於89.5t：至9〇.5它之間，李的熔 解溫度約於88.3^： i 89t：之間，而梅子與李子兩 者之溶點（melting p〇int)有著相當明顯地差異，因此 可用以分類鑑別梅與李。 再將兩者之核酸片段產物進行電泳分析（圖六 至圖八），可發現梅品系皆產生一個長度約％卟之 核酸片段’且李品系皆產生一個長度約U3bp之核 酸片段，完全符合當初設計引子之結果。由結果驗 證，以即時定量聚合酶連鎖反應偵測系統 (LlghtCycler System)及赛巴格林f光染齊h貞測樣 品之熔解溫度值，可快速精確地檢測鐘別梅品系與 李品系。本發明的其他特性將在下面詳細揭示且體 1323285 實施例觀察後變得顯而易見。 【實施方式】 本發明將詳細描述特殊的具體實施例。這些具體實 施例經由發明解釋提供，並非意欲用以限制本發 明。在發明的範圍及精神内，本發明存在傾向於包 括這些及其他變更及變動。 實施例一：DNA的萃取 依Lay etal., (2001)所發表之方法並作些許修 改，萃取梅子與李子葉片之DNA。秤取嫩葉0.1 克，於液態氮下磨成粉狀，加入1.0 mL萃取液（100 mM Tris-HCl，pH 8.0 ; 50 mM Na2EDTA，pH 8.0 ; 50 mM NaCl ; 10 mM b-mercapto-ethanol)及 0.1 mL 20% SDS，混合後置於65 °C水浴20分鐘，之後加 入0.5 mL 5 Μ醋酸奸激烈搖動後置入冰浴20分 鐘，反應完全後在4°C下以25000 X g離心20分 鐘，取上清液加入 1倍體積之 Isopropanol，於 -20°C 下放置 30〜60分鐘後，在 4°C 下以 12000X g離心15分鐘，取沉澱。沉澱物以0.5 mL 70%酒精洗去鹽類，之後將沉澱溶於300 pL之TE buffer (10 mM Tris-HCl，pH 8.0; 1 mM EDTA，pH 8.0) 1323285 並加入RnaseA (5 mg/mL)，置於室溫下1小時，加 入等體積的 phenol· chloroform -isoamyl alcohol ' (25 : 24 : 1)輕微搖動後，以12000 X g離心10分 鐘，重複此步驟兩次，取上清液加入兩倍體積無水 酒精及十分之一體積的NaOAc，置於-20°C下30 〜60分鐘後，以12000 X g離心15分鐘，取沉 澱，沉澱物以0.5 mL 70%酒精洗去鹽類，重複此步 驟兩次。DNA沉殿以Speed Vac乾燥後，溶於20 μί無菌水，在OD 260 nm下測其DNA濃度。 · 實施例二：DNA的定量 取1 pL的DNA溶液，溶於99 μι的去離子 水後，於分光光度計下測定Α260之吸光值後，計 算濃度（ng/pL)後，調整DNA濃度為100ng~L。 實施例三：聚合酶鏈鎖反應（PCR) ® 聚合酶鏈鎖反應試劑：1 X 緩衝溶液 (DyNAzymeTM II)，200 mM dNTP，0.5 mM 引子 (each)，1 unit DNA 聚合酶（DyNAzyme TM II) ’ 200 ng DNA模板，反應總體積為25 gL。而聚合酶鏈鎖 反應條件如下：97°C 3分鐘，再進行97°C 1秒、The sITS1 (SEQIDN04) and SITS2 (SEQID N05)' are shown in Figure 4. Figure 5 shows the sITS1 by the Instant Quantitative Polymerase Chain Reaction System (LightCycler System) and the Sai Binger fluorescent dye. And the melting temperature of the sITS2 primer to amplify the nucleic acid fragment product of Mei and the nucleic acid fragment of the plum and plum can be found to be quite obvious. The melting temperature of plum is about 89.5t: to 9〇. 5 Among them, the melting temperature of Li is about 88.3^: i 89t: between, and the melting point of plum and plum has quite obvious difference, so it can be used to classify plum and plum. The nucleic acid fragments of the two products were electrophoresed (Fig. 6 to Fig. 8), and it was found that the plum lines produced a nucleic acid fragment of about % ', and the Li line produced a nucleic acid fragment of about U3 bp in length, which was completely consistent with the original Design the results of the primer. The results were verified by the instant quantitative polymerase chain reaction detection system (LlghtCycler System) and the melting temperature value of the test sample, which can quickly and accurately detect the benphid and plum lines. Other features of the invention will be apparent from the following detailed description of the embodiment of the invention. [Embodiment] The present invention will be described in detail with particular embodiments. These specific examples are provided by way of illustration of the invention and are not intended to be limiting of the invention. The present invention is intended to embrace these and other modifications and variations within the scope and spirit of the invention. Example 1: Extraction of DNA The DNA of the leaves of plums and plums was extracted according to the method published by Lay et al., (2001). Weigh 0.1 g of young leaves, grind into powder under liquid nitrogen, and add 1.0 mL of extract (100 mM Tris-HCl, pH 8.0; 50 mM Na2EDTA, pH 8.0; 50 mM NaCl; 10 mM b-mercapto-ethanol) and 0.1 mL of 20% SDS, mixed and placed in a 65 °C water bath for 20 minutes, then added 0.5 mL of 5 Μ acetic acid and shaken vigorously and placed in an ice bath for 20 minutes. After the reaction was completed, centrifuge at 25000 X g for 20 minutes at 4 °C. The supernatant was added to 1 volume of Isopropanol, and left at -20 ° C for 30 to 60 minutes, and then centrifuged at 12000 X g for 15 minutes at 4 ° C to take a precipitate. The precipitate was washed with 0.5 mL of 70% alcohol, and then the precipitate was dissolved in 300 pL of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) 1323285 and RnaseA (5 mg/mL) was added. Place at room temperature for 1 hour, add an equal volume of phenol·chloroform -isoamyl alcohol ' (25 : 24 : 1), shake gently at 12000 x g for 10 minutes, repeat this step twice, and add the supernatant. Two volumes of absolute alcohol and one tenth of volume of NaOAc were placed at -20 ° C for 30 to 60 minutes, centrifuged at 12000 X g for 15 minutes, and the precipitate was taken. The precipitate was washed with 0.5 mL of 70% alcohol. , repeat this step twice. The DNA sink was dried with Speed Vac, dissolved in 20 μL of sterile water, and measured for DNA concentration at OD 260 nm. · Example 2: Quantification of DNA Take 1 pL of DNA solution, dissolve it in 99 μl of deionized water, and measure the absorbance of Α260 under a spectrophotometer. After calculating the concentration (ng/pL), adjust the DNA concentration to 100ng~L. Example 3: Polymerase chain reaction (PCR) ® polymerase chain reaction reagent: 1 X buffer solution (DyNAzymeTM II), 200 mM dNTP, 0.5 mM primer (each), 1 unit DNA polymerase (DyNAzyme TM II) ' 200 ng DNA template with a total reaction volume of 25 gL. The polymerase chain reaction conditions were as follows: 97 ° C for 3 minutes, and then 97 ° C for 1 second,
55°C 10秒、72°C 10秒反應，進行35次的DNA 12 1323285 增殖反應後，72°C 10分鐘。聚合酶鏈鎖反應產物以 2.0%瓊脂膠片進行電泳分析。 實施例四：電泳分析 利用DNA分子帶負電之特性，在通電之電泳 槽中，會依其分子形狀之大小或DNA片段之長 短，而影響其向正極之泳動率，使不同的DNA分 子在膠體（agarose)上分開，經漠化乙鍵（Ethidium Bromide，EtBr)染色後，可於紫外燈下觀察DNA 分子之存在。同時藉由DNA marker得知DNA分 子的長度大小，也可對DNA分子做概略之估量。 以2% agarose進行電泳分析，内含0.5 mg/ mL 的 EtBr (Ethidium Bromide)。取 DNA 樣品溶液， 加入 1/10 X 之追縱染劑（loading dye: 0.25°/〇 bromophenol blue, 0.25% xylene cyanol, 50% glycerol)，混合均勻，將其小心注入膠體之凹槽中， 電泳緩衝液為 lxTAE Buffer (0.04 M Tris base, 0.04 M acetate, 0.01 MEDTA);通入電壓 100 V，進行電 泳約25 min後，於紫外燈下觀察DNA之存在， 並拍照紀錄之。 實施例五：DNA定序 13 1323285 PCR擴增之基因產物委託台灣明欣生物科技 有限公司進行核酸序列分析。其DNA定序利用自動 定序儀 （ABI PRISM 377-96 DNA-Sequencer, Perkin-Elmer，CA，USA)，以試劑（ABI PRISM BigDye， Terminator cycler sequencing ready reaction kit, PE Applied Biosystem, USA)進行標定分析。 實施例六：即時定量聚合酶連鎖反應偵測系統 (LightCycler System)之進行 反應試劑：引子濃度為0.5 mM primer (each)， MgCl2 4 mM，DNA溶液2 μί，總體積為20 pL。即 時定量聚合酶連鎖反應（LightCycler PCR)條件如 下：95°C : 10分鐘，再進行95°C 0秒、53°C 5秒 及72°C 10秒反應，進行40次循環之DNA增值反 應，於反應完成後，升溫至95°C，再下降至65°C停 留30秒，而後再升溫至95°C，升溫過程中即時偵 測各樣品之螢光變化量，偵測速度為各樣品每0.1 秒偵測1次，所得結果進一步藉由儀器之電腦分析 以計算出熔解溫度，用以進行分析鑑定。 雖然本發明已以前述較佳實施例揭示，然其並 非用以限定本發明，任何熟習此技藝者，在不脫離 本發明之精神和範圍内，當可作各種之更動與修 改。如上述的解釋，關於加熱，機械破碎，酵素處 1323285 理及離心等等都可以作各種的修飾與變化，而不會 偏離本發明的精神及範圍。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。The reaction was carried out at 55 ° C for 10 seconds and 72 ° C for 10 seconds, and 35 times of DNA 12 1323285 was subjected to a proliferative reaction, followed by 72 ° C for 10 minutes. The polymerase chain reaction product was analyzed by electrophoresis on 2.0% agar film. Example 4: Electrophoresis analysis utilizes the negatively charged nature of DNA molecules. In the electrophoresis electrophoresis tank, depending on the size of the molecular shape or the length of the DNA fragment, the migration rate to the positive electrode is affected, so that different DNA molecules are in the colloid. Separate (agarose), after staining with Ethidium Bromide (EtBr), observe the presence of DNA molecules under UV light. At the same time, DNA strands can be used to estimate the length of DNA molecules, and DNA molecules can be roughly estimated. Electrophoresis analysis was performed with 2% agarose containing 0.5 mg/mL EtBr (Ethidium Bromide). Take the DNA sample solution, add 1/10 X of dyeing agent (loading dye: 0.25 ° / 〇 bromophenol blue, 0.25% xylene cyanol, 50% glycerol), mix well, carefully inject into the groove of the gel, electrophoresis The buffer was lxTAE Buffer (0.04 M Tris base, 0.04 M acetate, 0.01 MEDTA); the electrophoresis was carried out at a voltage of 100 V for about 25 min, and the presence of DNA was observed under an ultraviolet lamp and photographed. Example 5: DNA sequencing 13 1323285 The gene product of PCR amplification was entrusted to Taiwan Mingxin Biotechnology Co., Ltd. for nucleic acid sequence analysis. The DNA sequencing was performed using an automated sequencer (ABI PRISM 377-96 DNA-Sequencer, Perkin-Elmer, CA, USA) with reagents (ABI PRISM BigDye, Terminator cycler sequencing ready reaction kit, PE Applied Biosystem, USA). analysis. Example 6: Real-time quantitative polymerase chain reaction detection system (LightCycler System) reaction reagent: primer concentration of 0.5 mM primer (each), MgCl2 4 mM, DNA solution 2 μί, total volume of 20 pL. The conditions of the instant quantitative polymerase chain reaction (LightCycler PCR) were as follows: 95 ° C: 10 minutes, and then 95 ° C 0 seconds, 53 ° C 5 seconds and 72 ° C 10 seconds reaction, 40 cycles of DNA value-added reaction, After the reaction is completed, the temperature is raised to 95 ° C, and then lowered to 65 ° C for 30 seconds, and then heated to 95 ° C, and the amount of fluorescence change of each sample is detected immediately during the temperature rise, and the detection speed is for each sample. The detection was performed once in 0.1 seconds, and the obtained result was further analyzed by computer analysis to calculate the melting temperature for analysis and identification. While the present invention has been described in its preferred embodiments, it is not intended to limit the invention, and various modifications and changes can be made without departing from the spirit and scope of the invention. As explained above, various modifications and changes can be made to the heating, mechanical disruption, enzyme digestion, centrifugation, and the like without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims.
15 1323285 【圖示簡單說明】 圖一、真核核糖體DNA基因組成。 圖二、以ITS1 、ITS2及dITSI引子組對梅與李 DNA template進行擴增之電泳結果。15 1323285 [Simplified illustration] Figure 1. Composition of eukaryotic ribosomal DNA. Figure 2. Electrophoresis results of amplification of plum and plum DNA templates with ITS1, ITS2 and dITSI primers.
Lane 1 : 軟枝梅 Lane 7 : 山連梅 Lane 2 : 青廣梅 Lane 8 : 南田梅 Lane 3 : 胭脂梅 Lane 9 : 沙連李 Lane 4 : 尖形梅 Lane 10 · 狗屎李 Lane 5 : 大青梅 Lane 11 : 紅肉李 Lane 6 · 桃子梅 Lane 12 : 晚生桃接李 Lane M : pUC Mix Marker, 8 Lane 13 :水Lane 1 : Soft Branch Plum 7 : Shan Lianmei Lane 2 : Qing Guang Mei Lane 8 : Nan Tian Mei Lane 3 : Rouge Plum Lane 9 : Sha Lian Li Lane 4 : Pointed Plum Lane 10 · Shit Li Lan 5 : Large Ome Lin 11 : Red Meat Li Lane 6 · Peach Plum Lane 12 : Late Cherry Peach Lane M : pUC Mix Marker, 8 Lane 13 : Water
圖三、以ITS1 、ITS2及dITSI引子組對梅與李 DNA template進行擴增之電泳結果。Figure 3. Electrophoresis results of amplification of plum and plum DNA templates with ITS1, ITS2 and dITSI primers.
Lane 1 :信濃小梅 Lane 7 :石塔種梅Lane 1 : Shinano Xiaomei Lane 7 : Stone Tower Seed Plum
Lane 2 :豐後梅 Lane 8 :房柄雄梅Lane 2 : Feng Houmei Lane 8 : Fang Xiongmei
Lane 3 :胭脂梅（TARI) Lane 9 :凱爾西（Kelsey)李 Lane 4 :才兆幵j 梅 Lane 10 :陽金（Sungold)李Lane 3 : TARI Lane 9 : Kelsey Lee Lane 4 : Cai Zhao 幵 j Mei Lane 10 : Sungold Lee
Lane 5 :黃清海梅 Lane 11 :金冠（Goldenking)李 Lane 6 :萬山種梅 Lane 12 :金高（Gulfgold)李Lane 5 : Huang Qing Haimei Lane 11 : Golden Crown Lee Lane 6 : Wanshan Seed Plum Lane 12 : Gulf Gold Lee
Lane M : pUC Mix Marker, 8 Lane 13 :水 16 1323285 圖四、核糖體DNA上之ITS 1區域sITS 1及 sITS2反應引子組示意圖。 圖五、梅與李之melting temperature之測定。 圖六、以sITSl及sITS2引子組對梅DNA template進彳于擴增之電泳結果。Lane M : pUC Mix Marker, 8 Lane 13 : Water 16 1323285 Figure 4. Schematic diagram of the ITS 1 region sITS 1 and sITS2 reaction primer set on ribosomal DNA. Figure 5. Determination of the melting temperature of plum and plum. Figure 6. Electrophoresis results of the amplification of the plum DNA template with the sITS1 and sITS2 primers.
Lane 1 : 軟枝梅 Lane 5 ·* 大青梅 Lane 2 : 青廣梅 Lane 6 · 桃子梅 Lane 3 : 胭脂梅 Lane 7 : 山連梅 Lane 4 : 尖形梅 Lane 8 · 向田梅Lane 1 : Softwoods Lane 5 ·* Daqingmei Lane 2 : Qingguangmei Lane 6 · Taozimei Lane 3 : Rouge Plum Lane 7 : Shan Lianmei Lane 4 : Pointed Plum Lane 8 · Xiang Tianmei
Lane Μ ： pUC Mix Marker, 8 圖七、以sITSl及sITS2引子組對梅DNA template進行擴增之電泳結果。Lane Μ : pUC Mix Marker, 8 Figure 7. Electrophoresis results of amplification of plum DNA template with sITSl and sITS2 primers.
Lane 1 :信濃小梅 Lane 5 :黃清海梅Lane 1 : Shinano Xiaomei Lane 5 : Huang Qinghaimei
Lane 2 :豐後梅 Lane 6 :萬山種梅Lane 2 : Feng Houmei Lane 6 : Wanshan Seed Plum
Lane 3 :胭脂梅（TARI) Lane 7 :石塔種梅 Lane 4 :桃形梅 Lane 8 :房柄雄梅Lane 3 : TARI Lane 7 : Stone Pagoda Lane 4 : Peach Blossom Plum Lane 8 : House Handling
Lane Μ ： pUC Mix Marker, 8 圖八、以sITSl及sITS2引子組對李DNA template進彳亍擴增之電泳結果。Lane Μ : pUC Mix Marker, 8 Figure 8. Electrophoresis results of the amplification of the Li DNA template with the sITSl and sITS2 primers.
Lane 1 :沙連李 Lane 5 :凱爾西（Kelsey)李 17 1323285Lane 1 : Sha Lian Li Lane 5 : Kelsey Lee 17 1323285
Lane 2 :狗屎李 Lane 6 :陽金（Sungold)李Lane 2 : Shit Lee Lane 6 : Sungold Lee
Lane 3 :紅肉李 Lane 7 :金冠（Goldenking)李Lane 3 : Red Meat Lee Lane 7 : Golden Crown Lee
Lane 4 :晚生桃接李Lane 8 :金高（Gulfgold)李 Lane M : pUC Mix Marker, 8Lane 4 : Late Birthday Peach Lane 8 : Gulfgold Lee Lane M : pUC Mix Marker, 8
18 1323285 序列表 作卜月外修使)正本 <110>徐源泰 <120>以聚合酶鏈鎖反應鑑別梅及李之加工品 <140>092136093 <141>92/12/19 <160>5 <210>1 <211>15 <212>DNA <213>人工序列 <220> 係為進行PCR之引子 <223>位於植物rDNA ITS上， (primer) ° <400>1 gggggggttg cgttg 15 <210>2 <211>19 <212>DNA <213>人工序列 <220> <223>位於植物rDNA ITS上，将炎％ (primer) 〇 宁、马進行PCR之引子 <400>2 tccgtaggtg aacctgcgg 19 <210>3 <211>20 <212>DNA <213>人工序列 <220> <223>位於植物rDNA ITS上，信发☆ (primer)。 〜句進行PCR之引子 1323285 <400>3 gctgcgttct tcatcgatgc 20 <210>4 <211>22 <212>DNA <213>人工序列 <220> <223〉位於植物rDNA ITS上，係五、 引子（primer)。 、令進行 real-time PCR 之 <400>4 gacatatttg aagatgacga eg 22 <210>5 <211>18 <212>DNA <213>人工序列 <220>18 1323285 Sequence Listing for External Repair] Original <110> Xu Yuantai<120> Identification of processed products of plum and plum by polymerase chain reaction <140>092136093 <141>92/12/19 <160>5<210>1<211>15<212>DNA<213>Artificialsequence<220> is a primer for performing PCR<223> located on plant rDNA ITS, (primer) ° <400>1 gggggggttg cgttg 15 <210>2 <211>19 <212>DNA <213>Artificial sequence <220><223> is located on plant rDNA ITS, which is primer, The introduction of PCR by horses <400>2 tccgtaggtg aacctgcgg 19 <210>3 <211>20 <212>DNA <213> artificial sequence <220><223> is located on plant rDNA ITS, letter hair ☆ (primer). The PCR-inducing primer 1323285 <400>3 gctgcgttct tcatcgatgc 20 <210>4 <211>22 <212>DNA <213> artificial sequence <220><223> is located on the plant rDNA ITS, Department 5, primer. <400>4 gacatatttg aagatgacga eg 22 <210>5 <211>18 <212>DNA <213> artificial sequence <220>
<223>位於植物rDNA ITS上，係A 引子（primer)。 ' 崎進行 real-time PCR 之 <400>5 gggcgtacaa acgaacac 18<223> is located on the plant rDNA ITS and is a primer. ' Saki real-time PCR <400>5 gggcgtacaa acgaacac 18
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