TWI325058B - - Google Patents

Description

1325058 九、發明說明： 【發明所屬之技術領域】 本發明係相關於一種鑑別牛隻性別特異性序列及方 法。 【先前技術】 鳥類及哺乳動物動物之性別鑑別於經濟及研究發展比 十分重要。部分家禽家畜在其性成熟前或胚胎期甚至終生 之性別鑑定不易，但許多經濟活動，如買賣或飼養時烏類 及哺乳動物之公母比例，常需在性成熟前即需要鑑別知曉 該動物之性別。目前之技術方式，係有直接以外觀辨識、 核型（karyotype)、Barr小體（Barr b〇dy)或性別特異性抗體 測試（sex-specific antibody test)來分辨。但利用外觀辨識 的方法刀辨雌雄，常有判斷不易或誤判的情形丨另外，以 核型、Barr小體或雄性特異性抗體測試分辨的方式，其技 =層面％雜且成本提高，而所耗時間較長。击上所述，在 家禽家畜等經濟性動物的性別鑑定方自，仍缺少一種簡單 快速的鑑定方法。 【發明内容】 有名》司卜生別特異性序列（sex-specific sequence)』 動物之〖生染色體（sex chromosome)上，依動物性別 之不同而具有布勒 八啕及缺乏一特異性序列。 專有名詞『增Φ5古、+ a h方法』意指增幅核苷酸序列之試驗或 方法。例如，該&quot;增栌 万法包括聚合酶連鎖反應（PCR)試驗。 專有名詞『檢 聪』意指任何含有核酸之生物性物質之 1325058 較佳的是’該檢體為全血，血球、糞尿或***。 較佳的是，經SEQ ID NO: 4及SEQ ID NO: 5寡核苦 酸引子對所增幅之序列大小約為315bp;更佳的是，經 丨D NO:4及SEQ ID NO:5寡核苷酸引子對所增幅之序列為 SEQ ID NO. 3。 較佳的疋’經SEQ ID NO: 7及SEQ ID NO: 8募核苦 酸引子對所增幅之序列大小約為]5〇〇bp ;更佳的是，經 SEQ ID NO: 7及SEQ ID NO: 8寡核苷酸引子對所增幅之 序列為 SEQ ID NO. 6。 本發明另相關於一種鑑別牛隻性別特異性序列之寡核 苷酸引子對，其係一組或一組以上選自包含SEQ m N〇 4 與 SEQ ID NO. 5、及 SEQ ID Ν〇· 7 與 SEQ ID NO· 8 所組 成的的寡核苷酸引子對族群中，其中各組係包含兩條寡核 苷酸引子。 較佳的疋，各組养核苷酸引子對進而包括一内控制組 寡核苷酸引子對，·更佳的是’該内控制組寡核苷酸引子對 為 SEQ ID NO:1/SEQ ID N〇:2。 由本發明鑑別牛隻性別特異性序列及本發明所揭示之 方法，可提供一簡易的方式鑑定牛隻性別。 【實施方式】 t本發明係利用DNA指紋之技術，從具多態性之DNA 私紋％帶中’找到性別特異性之遺傳標記。而後將牛隻之 &amp;又DMA序列選殖出來’經序列分析後，與世界上前人已 發現之序列比對’發現均為前人所未發現之新序列。且根 出引至生別特異个生DNA(sex-specificDNA)自行設計 子對（phme「pak)，㈣所設計之引子料行增幅反應 及電泳分析，經電、灰 ,^片上之DNA分子分佈，可準確判斷 出動物之性別。 下列貫施例用於示範說明本發明。這些實施例不以任 何方式意欲限制本發明的範圍Μ旦用以指示如何實施本發 明的材料與方法。 之方法1325058 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for identifying a sex-specific sequence and method for a cow. [Prior Art] Gender discrimination between birds and mammalian animals is important in economic and research development ratios. Some poultry livestock are not easy to identify before sex or during embryonic or even life, but many economic activities, such as the proportion of males and females in blacks and mammals when buying or selling, often need to identify the animal before sexual maturity. Gender. The current technical approach is directly distinguished by appearance recognition, karyotype, Barr b〇dy or sex-specific antibody test. However, the method of recognizing the appearance of the knife is used to distinguish between male and female, and it is often difficult to judge or misjudge. In addition, the method of karyotype, Barr, or male-specific antibody test and resolution is mixed and the cost is increased. It takes a long time. According to the above, the gender identification of economic animals such as poultry and livestock is still lacking a simple and rapid identification method. SUMMARY OF THE INVENTION The well-known "sex-specific sequence" of the animal's sex chromosome has a Buhler gossip and lacks a specific sequence depending on the sex of the animal. The proper noun "increasing Φ5 ancient, + a h method" means a test or method for increasing the nucleotide sequence. For example, the &quot;enrichment method includes a polymerase chain reaction (PCR) test. The proper term "detection" means any biological substance containing nucleic acid 1325058. Preferably, the sample is whole blood, blood cells, feces or semen. Preferably, the amplified sequence of the oligo-nucleotide primer pair of SEQ ID NO: 4 and SEQ ID NO: 5 is about 315 bp; more preferably, 丨D NO: 4 and SEQ ID NO: 5 The sequence of the amplification of the nucleotide primer pair is SEQ ID NO. Preferably, the size of the amplified nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 is about 5 bp; more preferably, SEQ ID NO: 7 and SEQ ID The sequence in which the NO:8 oligonucleotide primer pair is amplified is SEQ ID NO. The invention further relates to an oligonucleotide primer pair for identifying a sex-specific sequence of a bovine, which is selected from the group consisting of SEQ m N〇4 and SEQ ID NO. 5, and SEQ ID Ν〇. 7 In the group of oligonucleotide primer pairs consisting of SEQ ID NO. 8, wherein each group comprises two oligonucleotide primers. Preferably, each group of nucleotide promoter pairs further comprises an internal control group oligonucleotide primer pair, and more preferably, the internal control group oligonucleotide primer pair is SEQ ID NO: 1 / SEQ ID N〇: 2. The identification of bovine sex-specific sequences by the present invention and the methods disclosed herein provide an easy way to identify the sex of a cow. [Embodiment] The present invention uses a technique of DNA fingerprinting to find a gender-specific genetic marker from a polymorphic DNA pattern. Then the DMA sequence of the cattle &amp; </ br> was selected and 'sequence analysis, and the sequence sequence found by the world's predecessors' was found to be a new sequence not found by the predecessors. And roots are introduced to the bio-specific DNA (sex-specific DNA) self-designed pair (phme "pak", (d) designed by the primers line amplification reaction and electrophoresis analysis, the distribution of DNA molecules on electricity, ash, ^ tablets The following examples are intended to illustrate the invention. The examples are not intended to limit the scope of the invention in any way to indicate how to practice the materials and methods of the invention.

檢體 自動物體採取血液樣本，將所採得之血液樣本收集在 一含有抗凝血劑的試管内。Samples Automated objects take blood samples and collect the collected blood samples in a test tube containing anticoagulant.

1.2抽取DNA 採集後之血液以Shiau and Huang (1997)所建議之方 式萃取出基因組DNA ( genomic DNA)。基因組D N A 經定量後，以滅菌之去離子水WDNA稀釋成5 〇ng/&quot;卜 供進行增幅試驗之用。 1.3增幅試驗 增幅試驗係參照Sambrook等人（1989)所建議之方法 修改而來。增幅試驗之各反應物濃度如下：1〇mM TrisHC丨 ρΗ8·0，1_5mM MgCI2 ’ 50mM KC卜各 i〇〇m M 之 dATp、 dCTP、dGTP 及 dTTP ’ 各 0.14&quot; M 之個別引子，1〇〇ng 的基因組DNA、0.5U Dynazyme丨丨聚合酶（Fjnnzymes 〇y) ’而後加滅菌之去離子水2 y卜使每個試管内之反應體 積為25〆丨。所使用之引子對為逄機序列引子對 1325058 (Operon,USA)。 所進行之增幅試驗為RAPD_pcR(rar)d〇ni amplified polymorphic DNA PCR)之反應條件為：94〇c變性5分鐘，而 後母個增幅反應進行45個溫度週期（94°c變性1分鐘，36 C進行引子對黏合1分鐘及72°C延長2分鐘），再經72°C 延長7分鐘使DNA完全成為雙股。 1.4 DNA產物之分離與回收 將PCR產物之少量（每個2〇 v丨），加入適當指示劑後， 鲁 於2.〇。/。瓊脂糖凝膠50伏特的電壓下進行電泳。電泳後， 將凝膠染以溴化乙啶（ethidium bromide)並於UV光源下觀 察’發現可於雌雄動物之基因組DNA增幅出一段可分辨不 同性別之特異性片段’切下此含有特異性DNA片段之凝 膠’加以回收純化，並將已純化之DNA片段吾以原逢機引 子再次進行PCR。 ,1.5勝任細胞之備製 取30 ml經37°C振盪培養至OD6q〇= 0.4〜0.6之大腸 鲁 桿菌菌液，於4°C下以6,000 rpm離心5分鐘，去上層液， 加入15 ml冰冷之50 mM CaCl2重懸浮菌體，冰浴10分 鐘。再於4。〇下以6,000 rpm離心5分鐘，去上層液，加入 15 ml冰冷之50 mM CaCl2溶液，冰浴1 〇分鐘。重覆上 述步驟，並以相同條件離心後，細胞重懸浮於1 ml 50 mΜ CaCl2中，再加入總體積十分之一的DMSO後，於-7〇°C中 貯存備用。 · 1·6 轉形反應（transformation) 1325058 根據1.4之方法所得之產物，參照Invitrogen操作手 冊，取 2//丨經二次 PCR 之產物，與 1从丨 pcDNA3.1/V5-His-TOPO (Cat. No. K4800-0” 載體戍 pCRII-TOPO ( Cat. No. K4600-01 )載體， (Invitrogen,CA，USA)混合。操作方法如下：根據ΤΟΡΟ ΤΑ1.2 Extraction of DNA The collected blood was extracted from genomic DNA as suggested by Shiau and Huang (1997). After the genomic D N A was quantified, it was diluted with sterile sterilized deionized water WDNA into 5 〇ng/&quot; for the amplification test. 1.3 Amplification test The amplification test was modified by the method recommended by Sambrook et al. (1989). The concentrations of the respective reactants in the amplification test were as follows: 1 mM TrisHC丨ρΗ8·0, 1_5 mM MgCI2 '50 mM KC, dATp, dCTP, dGTP and dTTP of each 0.1 M&lt; M individual primer, 1〇 〇ng genomic DNA, 0.5 U Dynazyme 丨丨 polymerase (Fjnnzymes 〇y) ' and then sterilized deionized water 2 y to make the reaction volume in each tube is 25 〆丨. The primer pair used is the primer sequence primer pair 1325058 (Operon, USA). The amplification test carried out was RAPD_pcR(rar) d〇ni amplified polymorphic DNA PCR). The reaction conditions were: 94〇c denaturation for 5 minutes, and the latter parental amplification reaction was carried out for 45 temperature cycles (94°c denaturation for 1 minute, 36 C). The primers were ligated for 1 minute and extended at 72 ° C for 2 minutes, and then extended by 72 ° C for 7 minutes to completely double the DNA. 1.4 Isolation and recovery of DNA products A small amount of PCR product (each 2〇 v丨) is added to the appropriate indicator and is then 2. /. Electrophoresis was carried out at a voltage of 50 volts on an agarose gel. After electrophoresis, the gel was stained with ethidium bromide and observed under UV light source. 'It was found that the genomic DNA of male and female animals could be amplified by a specific fragment that can distinguish different sexes'. The gel of the fragment was recovered and purified, and the purified DNA fragment was subjected to PCR again using the original primer. , 1.5 Compatible for the preparation of cells. Prepare 30 ml of E. coli bacteria cultured at 37 ° C with shaking to OD6q〇 = 0.4~0.6, centrifuge at 6,000 rpm for 5 minutes at 4 ° C, remove the supernatant, add 15 ml of ice cold. The cells were resuspended in 50 mM CaCl2 and ice bathed for 10 minutes. Then again 4. Centrifuge at 6,000 rpm for 5 minutes, remove the supernatant, add 15 ml of ice-cold 50 mM CaCl2 solution, and ice bath for 1 minute. After repeating the above procedure and centrifuging under the same conditions, the cells were resuspended in 1 ml of 50 m Μ CaCl 2 , and then added to one tenth of the total volume of DMSO, and stored at -7 ° C for use. · 1·6 transformation reaction 1325058 According to the method of 1.4, refer to the Invitrogen operating manual, take 2 / / 丨 through the secondary PCR product, and 1 from 丨pcDNA3.1/V5-His-TOPO ( Cat. No. K4800-0" carrier 戍pCRII-TOPO (Cat. No. K4600-01) carrier, (Invitrogen, CA, USA) was mixed. The method of operation is as follows: according to ΤΟΡΟ ΤΑ

Cloning® Kit ( Invitrogen®)手冊所述’依次加入經電泳確 認無誤之PCR產物及0.5以丨pCRΗ- ΤΟΡΟ®載體，調整 反應總體積為5以丨’於室溫下作用5分鐘’再移至冰上備 用’以進行下一步之轉形。添加約50 ng〜1 eg重組質體 D N A或黏接反應後之D N A，加入1.5之方法所製備之勝任 細胞1 00 # I ’冰浴1小時後，於42Ό水浴進行熱休克，加 入0.9 ml LB培養液，於37°C下水浴1小時，取1 〇〇〜200 // I菌液’以曲型玻璃均勻塗佈選擇性培養基，經37。〇培 養1 2〜16小時即可。 轉形反應所獲得的菌落經Cracking method(林，1991) 初步篩選，挑出可能菌落再經培養後，以質體DΝΑ萃取套 組（QIAprep Spin Miniprep kit, Cat. No. 27106, QIAGEN) 來分離DNA，所得之DNA調整其濃度為〇.5y g//i卜以 M13 順向引子 /逆向引子，及 ab| Prism Big Dye Terminator Cycle 定序反應套組（BigDye v 3」；perkin_E|mer/AB，L〇tIn the Cloning® Kit (Invitrogen®) manual, 'in turn, add the PCR product confirmed by electrophoresis and 0.5 丨pCRΗ-ΤΟΡΟ® carrier, adjust the total volume of the reaction to 5 丨' at room temperature for 5 minutes' and then move to Spare on ice 'for the next turn. Add about 50 ng~1 eg of recombinant plastid DNA or DNA after the adhesion reaction, add the competent cells prepared by the method of 1.5 to 1 00 # I 'ice bath for 1 hour, heat shock in 42 Ό water bath, add 0.9 ml LB The culture solution was immersed in a water bath at 37 ° C for 1 hour, and 1 〇〇~200 // I bacteria solution was uniformly coated with a selective medium in a curved glass, and 37 was passed. 〇培养1 2~16 hours. The colonies obtained by the transformation reaction were initially screened by the Cracking method (Lin, 1991), and the possible colonies were picked and cultured, and then separated by a QDprep Spin Miniprep kit (Cat. No. 27106, QIAGEN). DNA, the resulting DNA was adjusted to a concentration of 〇.5y g//i to M13 forward primer/reverse primer, and ab| Prism Big Dye Terminator Cycle sequencing reaction set (BigDye v 3); perkin_E|mer/AB , L〇t

No. 0303013) ’ 在 Applied Biosystem 3100 DNA 自動定 序儀（Perkin-Elmer Cetus Corp·, Forster City, CA, USA) 上進行定序所分離之D N A序列。 1 -6引子之設計及性別特異性序列之增幅反應 10 1325058 根據定序後之DNA序列’設計可鑑定不同動物性別之 引子對。No. 0303013) ' The sequenced D N A sequence was sequenced on an Applied Biosystem 3100 DNA Automated Sequencer (Perkin-Elmer Cetus Corp., Forster City, CA, USA). Design of 1 -6 primers and amplification response of sex-specific sequences 10 1325058 Based on the sequenced DNA sequence design, primer pairs for different animal sexes can be identified.

取動物體基因組DNA(50ng/#丨），以下述之可鑑定不同 動物性別之引子對進行增幅反應試驗。同時，根據1 8 S rRNA序列設計之引子對18S-F/18S-R (Hedges等人，1990) 同時進行增幅試驗，作為增幅試驗之内控制組。 18S-F(SEQ ID NO:1): 5，-AGCTCTTTCT CGATTCCGTG-3;The animal genomic DNA (50 ng/#丨) was taken and subjected to an amplification reaction test using the following primer pairs which can identify the sexes of different animals. At the same time, 18S-F/18S-R (Hedges et al., 1990) was simultaneously subjected to an amplification test based on the primer design of the 18S rRNA sequence as an internal control group for the amplification test. 18S-F (SEQ ID NO: 1): 5, -AGCTCTTTCT CGATTCCGTG-3;

18S-R(SEQ ID NO:2): 5'-GGGTAGACAC AAGCTGAGCC-3 18S-F/18S-R(SEQ ID NO:1/SEQ ID NO:2)引子對之 增幅反應條件如下：94t變性5分鐘，而後每個増幅反應進 行30個溫度週期（94°C變性1分鐘，58。(：進行引子對黏合 45秒，及72°C延長1分鐘），再經72。(：延長7分鐘使DNA 完全成為雙股。 實施例二：水牛性別特显袖年 2.1台灣水牛6Ma//_s)之性別特異性序列 根據實施例一之1.彳至彳_4所述之方法，得第一圖所示 之RAPD-PCR電泳膠圖，且發現於雄性台灣水牛之dna 片段分布圖上’具有一性別特異性序歹，如箭頭標示處。 純化該性別特異性序列，並根據14之操作方法將該序列 嵌入_3.1/ν5·Η丨s_T〇p〇載體，形成—質體DN:(第 -圖）。質體DNA經轉形反應後，㈣體DNa，並定序台 灣水牛的性別特異性序列SEQ |D盼3。根據MO ^ 1325058 N 〇: 3 ’設計用以增幅台灣水牛性別特異性序列之寡核苷酸 引子對為18S-R (SEQ ID NO: 2): 5'-GGGTAGACAC AAGCTGAGCC-3 18S-F/18S-R (SEQ ID NO: 1 / SEQ ID NO: 2) The amplification reaction conditions of the primer pair are as follows: 94t denaturation for 5 minutes Then, each of the sputum reactions was carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, 58. (: primer pair for 45 seconds, and 72 ° C for 1 minute), and then 72. (: 7 minutes extension of DNA It is completely double-stranded. Example 2: Buffalo Sexual Sleeve Year 2.1 Taiwan Buffalo 6Ma//s) Gender-specific sequence According to the method described in Example 1, 彳 to 彳 _4, the first figure is obtained. The RAPD-PCR electrophoresis map is shown and found on the distribution map of male Taiwan buffalo DNA fragments with a gender-specific sequence, as indicated by the arrow. Purify the sex-specific sequence and according to the method of 14 The sequence is embedded in the _3.1/ν5·Η丨s_T〇p〇 vector to form the plastid DN: (Fig.). After the transformation of the plastid DNA, (4) the body DNa, and sequence the sex-specific sequence of the Taiwan buffalo SEQ | D is expected to be 3. According to MO ^ 1325058 N 〇: 3 'The oligonucleotide primer pair designed to increase the sex-specific sequence of Taiwan buffalo is

BuSexOPC16F (SEQ ID NO:4): 5’-CACTCCAGTC TAACACAGTC AGTAG-3， BuSexOPC16R (SEQ ID NO:5): 5’-CTCCAGAGAC TGCATGCTAT GGTGA-31BuSexOPC16F (SEQ ID NO: 4): 5'-CACTCCAGTC TAACACAGTC AGTAG-3, BuSexOPC16R (SEQ ID NO: 5): 5'-CTCCAGAGAC TGCATGCTAT GGTGA-31

取上述引子對與台灣水牛之基因組DNA(50ng/# I)進 行增幅試驗’增幅試驗條件如下：94°c變性5分鐘，而後每 個増幅反應進行35個溫度週期（94°C變性1分鐘，661進 行引子對黏合45秒’及72〇C延長1分30秒），再經72°C 延長7分鐘使DNA完全成為雙股。進行電泳分析，結果見 第三圖所示。 由第二圖的結果，第1列為1 〇〇bp之分子標記，第2·6 列為雌性台灣水牛之增幅結果，第7_彳彳列為雄性台灣水牛 之增幅結果。由電泳膠圖可發現本發明之募核苷酸引子對The above primers were used to increase the amplitude of the genomic DNA (50 ng/# I) of Taiwan buffalo. The conditions of the amplification test were as follows: denaturation at 94 ° C for 5 minutes, and then each of the amplitude reaction was carried out for 35 temperature cycles (denaturation at 94 ° C for 1 minute, 661 was subjected to primer pair bonding for 45 seconds ' and 72 ° C for 1 minute and 30 seconds, and then extended by 72 ° C for 7 minutes to completely double the DNA. The electrophoresis analysis was carried out, and the results are shown in the third figure. From the results of the second figure, the first column is the molecular marker of 1 〇〇 bp, the second column is the result of the increase of the female Taiwan buffalo, and the seventh column is the result of the increase of the male Taiwan buffalo. The nucleotide primer pair of the present invention can be found from the electrophoresis gel map

BuSexOPC16F/ BuSexOPC16R (SEQ ID NO:4/SEQ ID N〇:5)，於箭頭標示315bp處，發現在雄性水牛基因組 DNA，可專一性增幅性別特異性序列，且其中每一基因組 DNA均可增幅256bp之18「RNA内控制組片段（箭頭標示BuSexOPC16F/ BuSexOPC16R (SEQ ID NO: 4/SEQ ID N〇: 5), found at 315 bp in the arrow, found in male buffalo genomic DNA, can specifically increase the sex-specific sequence, and each of the genomic DNA can increase by 256bp 18" RNA internal control group fragment (arrow mark

18S 者），故 BuSex〇PC16F/ BuSexOPC16R (SEQ ID NO:4/SEQ ID NO:5)確實可提供快速及準確之性別鑑定方 法。 2 _ 2不同品種之牛隻性別鑑定 取水牛（B)、黃牛（γ)、荷蘭牛（η)、安格斯牛（八）及海弗 12 1325058 牛（HF)等不同品種之公母牛隻檢體，同時以 BuSexOPC16F/ BuSexOPCI 6R (SEQ ID NO:4/SEQ ID NO:5)引子對，進行增幅試驗，得第四圖所示之膠圖。由第18S), therefore BuSex〇PC16F/ BuSexOPC16R (SEQ ID NO:4/SEQ ID NO:5) does provide a fast and accurate method of gender identification. 2 _ 2 Different breeds of cattle were identified for different breeds of buffalo such as buffalo (B), yellow cattle (γ), Dutch cattle (η), Angus cattle (eight) and Haifu 12 1325058 cattle (HF). Only the sample was simultaneously subjected to an amplification test using the BuSexOPC16F/BuSexOPCI 6R (SEQ ID NO: 4/SEQ ID NO: 5) primer pair to obtain the gel pattern shown in the fourth figure. By the first

四圖結果發現 BuSexOPC16F/ BuSex〇PC16R (SEQ ID NO:4/SEQ ID NO:5)引子對皆可於各品種之雄性牛隻增幅 一如箭頭標示之 315bp片段，故 BuSexOPC16F/ BuSexOPC16R (SEQ ID NO:4/SEQ ID NO:5)引子對，確可 應用在不同品種之牛隻的性別鑑定。 • 實施例三：黃牛性別特異性戽列 3.1黃牛（Sos /7?cy/ct/s)性別特異性序列 根據實施例一之1 · 1至1.4所述之方法，得第五圖所示 之RAPD-PCR電泳膠圖，且發現於雄性黃牛之DNA片段分 布圖上’具有一性別特異性序列’如箭頭標示處。純化該 性別特異性序列，並根據1 ·4之操作方法，將該序列喪入 PCRM-TOPO載體，形成一質體DNA(第六圖）。質體Dna 經轉形反應後，抽質體DNA，並定序黃牛的性別特異性序 ® 列，如SEQ ID NO:6所列。根據SEQ ID NO:6，設計用以 增幅黃牛性別特異性序列之寡核苷酸引子對為 YCaOPAG06F(SEQ ID NO:7): 5'-TCCTCCTGCA ATGTGTGAGA CCTG-31 YCaOPAG06R(SEQ ID NO:8): 5，-CAAGATTCCA AGGCTGCAAC AGCG二3’ 上述引子對與黃牛之之基因組DNA(50ng///|)進行増 幅試驗’增幅試驗條件如下：94°C變性5分鐘，而後每個増The results of the four images show that the BuSexOPC16F/BuSex〇PC16R (SEQ ID NO:4/SEQ ID NO:5) primer pairs can be increased in the male cattle of each species as the 315 bp fragment indicated by the arrow, so BuSexOPC16F/ BuSexOPC16R (SEQ ID NO) :4/SEQ ID NO: 5) The primer pair can be applied to the sex identification of different breeds of cattle. • Example 3: Yellow cattle sex-specificity 3.1 3.1 Yellow cattle (Sos / 7? cy / ct / s) gender-specific sequence according to the method described in Example 1 1 · 1.4 to obtain the fifth figure RAPD-PCR electrophoresis gel map, and found on the DNA fragment distribution map of male cattle with 'sex-specific sequence' as indicated by the arrow. The sex-specific sequence was purified and the sequence was sacrificed into the PCRM-TOPO vector according to the protocol of 1-4 to form a plastid DNA (sixth image). After the plastid DNA has been transformed, the plastid DNA is extracted and the sex-specific sequence of the yellow cattle is sequenced as listed in SEQ ID NO: 6. According to SEQ ID NO: 6, an oligonucleotide primer pair designed to amplify a sex-specific sequence of a yellow cattle is YCaOPAG06F (SEQ ID NO: 7): 5'-TCCTCCTGCA ATGTGTGAGA CCTG-31 YCaOPAG06R (SEQ ID NO: 8): 5,-CAAGATTCCA AGGCTGCAAC AGCG 2 3' The above primers were used to test the amplitude of the genomic DNA (50 ng///|) with the yellow cattle. The increase test conditions were as follows: denaturation at 94 ° C for 5 minutes, then each 増

13 幅反應進行25個溫度週期(94t變性，分鐘，抓進行引 2黏合1分鐘’ &amp; 72°C延長1分鐘），再經饥延長7 刀釦使DNA凡全成為雙股。進行電泳分析，結果見第七圖 所示。 由第七圖的結果，第1列為100bp之分子標記，第2·6 列為雌性黃牛之增幅結果，第7-11列為雄性黃牛之增幅結 果。由電泳膠圖可發現本發明之寡核苦酸引子對The 13 reactions were carried out for 25 temperature cycles (94t denaturation, minute, grasping, 2 bonding, 1 minute' &amp; 72 °C for 1 minute), and then hunger-extending 7 knives to make the DNA all double. The electrophoresis analysis was carried out, and the results are shown in the seventh figure. From the results of the seventh graph, the first column is the molecular marker of 100 bp, the second column is the increase result of the female ox, and the seventh column is the increase result of the male ox. The oligonucleotide primer pair of the present invention can be found by electrophoresis gel

VCaOPAG06F/ YCaOPAG〇6R(SEQ ID N0:7/SEQ |D 盼8)，於箭頭標示約[⑽處，可以增幅雄性黃牛的基因 組DNA中的㈣特異性序列’且其中每—基因組嶋均可 增幅256bp之，8SrRNA内控制組片段，可以證明本發明之 性別特異性引子對SEQ ID N〇：7/SEQ ID N0:8，可以專一 性增幅雄性黃牛的基因組DNA中的性別特異性序列，快速 及準確提供之性別鐘定方法。 3.2不同品種之牛隻性別鋥定 取黃牛（Y)、荷蘭牛（H)、安格斯牛（A)、海弗牛（HF)及 台灣水牛⑻等不@品種之公母牛數隻之檢體，以 YCBOPAG06F/ YC3OPAG06R (SEQ ,D NO：7/SEQ ID N0.8矧子對，進行性料異性序狀增 八圖所示^圖可以得知SEQ丨讀:7/啦=〇第8 引子對，發現可於荷蘭牛、安格斯牛及海弗牛之基因組隱 亦可增幅該約Wb大小的性別特異性片段故VCaOPAG06F/YCaOPAG〇6R (SEQ ID NO: 7/SEQ | D 8), indicated by the arrow [(10), can increase the (four) specific sequence in the genomic DNA of male cattle] and each of the genomes can increase 256 bp, 8SrRNA internal control group fragment, can prove that the sex-specific primer of the present invention can specifically increase the sex-specific sequence in the genomic DNA of male yellow cattle by SEQ ID N〇: 7/SEQ ID NO: 8 Accurately provide a gender-determining method. 3.2 The sex of different breeds of cattle is determined by the number of males of the breeds such as yellow cattle (Y), Dutch cattle (H), Angus cattle (A), Haifu cattle (HF) and Taiwan buffalo (8). The sample is YCBOPAG06F/YC3OPAG06R (SEQ, D NO:7/SEQ ID N0.8 scorpion pair, and the sequence of the anisotropy of the material is shown in the figure of 8). The SEQ 丨 reads: 7/啦=〇 8 primer pairs, found that the genome of the Dutch cattle, Angus cattle and Haifu cattle can also increase the size of the Wb-specific gender-specific fragments

YCaoPAG06R 牛女格斯牛及海弗牛的性別鑑定。 · 由上所述，本發明之鑑別牛 及其性別特異性序列，確具上述的功:特而異=的方法 作的不同修正及變化對於熟習該項技：者而而=發明可 會偏離本發明的範圍 °々顯然不 的偏好具體事實，必須；;的？：本發明已經敘述於特定 义肩了解的是本發明不應被不 於該等特定的偏好具體事實 、制 已述模式方面，對於熟習該項技術者而言，顯而易知修正 亦被涵蓋於下列申請專利範圍之内。 / 【圖式簡單說明】 第-圖係為水牛基因組嶋經逢機弓丨子對進行增 驗之電泳膠圖。 第二圖係為水牛特異性序列嵌合至載體之基因圖譜。 第三圖係為以SEQ ID N0:4/SEQ ID N〇:5增幅水牛基 因組DNA之電泳膠圖。 土 第四圖係為以SEQ ID N0:4/SEQ丨D N〇:5增幅各不同 品種牛隻基因組D N A。 第五圖係為黃牛基因組DNA經逢機弓丨子對進行增幅試 驗之電泳膠圖。 第/、圖係為黃牛特異性序列嵌合至載體之基因圖譜。 第七圖係為以SEQ ID N0:7/SEQ丨〇 ν〇·8增幅黃牛基 因組DNA之電泳膠圖。 第八圖係為以SEQ ID N0:7/SEQ ID N〇:8增幅各不同 品種牛隻基因組DNA。 【主要元件符號說明】 無 1325058 參考文件：YCaoPAG06R Sex identification of cattle female Gus cattle and Haifu cattle. · As described above, the identification of cattle and their gender-specific sequences of the present invention does have the above-mentioned work: different modifications and changes made by the method of the specific method are familiar to the skill: The scope of the invention 々 obviously does not favor the specific facts that must be; The present invention has been described in terms of specific definitions. It is to be understood that the present invention should not be deviated from the specific facts of the particular preferences, and the mode of the described modes. For those skilled in the art, it is obvious that the correction is also covered. It is within the scope of the following patent application. / [Simple description of the diagram] The first image is an electrophoresis gel image of the buffalo genome. The second panel is a genetic map of the buffalo-specific sequence chimeric to the vector. The third panel is an electrophoresis gel of the Buffalo genomic DNA amplified with SEQ ID NO: 4/SEQ ID N::5. The fourth map is the genomic D N A of each of the different breeds with SEQ ID N0:4/SEQ丨D N〇:5. The fifth picture is an electrophoresis gel image of the genomic DNA of the yellow cattle subjected to an increase in the amplitude of the genomic DNA. The / map is a genetic map of the bovine specific sequence chimeric to the vector. The seventh panel is an electrophoresis gel of the yellow genomic DNA of the SEQ ID NO: 7/SEQ 丨〇 ν. The eighth figure shows the increase in genomic DNA of each of the different breeds of SEQ ID NO: 7 / SEQ ID N: 8 . [Main component symbol description] None 1325058 Reference file:

1. Shiau, J.W. &amp; Huang, M.C. (1997) The probe of repetitive sequence for DNA fingerprinting in Holstein cattle. Journal of the Agriculture Association of China, 177: 1-10.1. Shiau, J.W. &amp; Huang, M.C. (1997) The probe of repetitive sequence for DNA fingerprinting in Holstein cattle. Journal of the Agriculture Association of China, 177: 1-10.

2. Sambrook, J., E. F. Fritsch &amp; T. Maniatis. 1989. In vitro amplification of DNA by the polymerase chain reaction, chapter 14. In: Molecular cloning: A Labortory Manual, ed. by Cold Spring Harbor Labortory Press. Cold Spring Harbor New York. 3. Hedges, S.B., Moberg, K.D. &amp; Maxson, L.R. (1990) Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships. Molecular Biology and Evolution, 7:607-633.2. Sambrook, J., EF Fritsch &amp; T. Maniatis. 1989. In vitro amplification of DNA by the polymerase chain reaction, chapter 14. In: Molecular cloning: A Labortory Manual, ed. by Cold Spring Harbor Labortory Press. Cold Spring Harbor New York. 3. Hedges, SB, Moberg, KD &amp; Maxson, LR (1990) Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships. Molecular Biology and Evolution, 7:607 -633.

4 ·林志生，1991。自牛及山羊基因組集合庫中選殖酪蛋白基因。中興大學碩士論文。 1325058 序列表 &lt;11〇&gt;行政院農業委員會 &lt;120〉鑑別牛隻酬之特異性序列及方法 &lt;130&gt; &lt;160〉 8 &lt;170&gt; Patentln version 3.34 · Lin Zhisheng, 1991. The casein gene was selected from a pool of cattle and goat genomes. Master's thesis of Zhongxing University. 1325058 Sequence Listing &lt;11〇&gt; Executive Yuan Agricultural Committee &lt;120>Specific sequence and method for identifying cattle rewards &lt;130&gt;&lt;160〉 8 &lt;170&gt; Patentln version 3.3

&lt;210&gt; 1 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; 18SrRNA &lt;400&gt; 1 agctctttct cgattccgtg &lt;210&gt; 2 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; 18SrRNA &lt;400&gt; 2 gggtagacac aagctgagcc 20 20&lt;210&gt; 1 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; 18SrRNA &lt;400&gt; 1 agctctttct cgattccgtg &lt;210&gt; 2 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; 18SrRNA &lt;400&gt;; 2 gggtagacac aagctgagcc 20 20

&lt;210〉 3 &lt;211&gt; 321 &lt;212&gt; DNA &lt;213&gt; Bubalus bubalis &lt;400&gt; 3 cacactccag tctaacacag tcagtagatt atcatcatcc aatattttaa ctgtggaaca ttccacaagt cttcttcatg cacaccatgt caaacatacc cagtagctta tcaccatccc gtgtttcagt gcagtgacat gattcaagtg tttcttcatt ttctgcaggt aaacacactc aatagtatgt caccaatgtt tcacctgtga catgattcct cttaatttat ttagttggct gtgttgggtc atagttgtgt cacataagat agtcctcgag acaagccaac tctcaccata gcatgcagtc tctggagtgt g 60 120 180 240 300 321&Lt; 210> 3 &lt; 211 &gt; 321 &lt; 212 &gt; DNA &lt; 213 &gt; Bubalus bubalis &lt; 400 &gt; 3 cacactccag tctaacacag tcagtagatt atcatcatcc aatattttaa ctgtggaaca ttccacaagt cttcttcatg cacaccatgt caaacatacc cagtagctta tcaccatccc gtgtttcagt gcagtgacat gattcaagtg tttcttcatt ttctgcaggt aaacacactc aatagtatgt caccaatgtt tcacctgtga catgattcct cttaatttat ttagttggct Gtgttgggtc atagttgtgt cacataagat agtcctcgag acaagccaac tctcaccata gcatgcagtc tctggagtgt g 60 120 180 240 300 321

&lt;210&gt; 4 &lt;211&gt; 25 &lt;212&gt; DNA l 1325058 &lt;213&gt; Bubalus bubalis &lt;400&gt; 4 cactccagtc taacacagtc agtag 25 &lt;210&gt; 5 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; Bubalus bubalis &lt;400&gt; 5 ctccagagac tgcatgctat ggtga 25 &lt;210&gt; 6 &lt;211&gt; 1529 &lt;212&gt; DNA &lt;213&gt; Bos indicus &lt;400&gt; 6&lt;210&gt; 4 &lt;211&gt; 25 &lt;212&gt; DNA l 1325058 &lt;213&gt; Bubalus bubalis &lt;400&gt; 4 cactccagtc taacacagtc agtag 25 &lt;210&gt; 5 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; Bubalus bubalis &lt;400&gt; 5 ctccagagac tgcatgctat ggtga 25 &lt;210&gt; 6 &lt;211&gt; 1529 &lt;212&gt; DNA &lt;213&gt; Bos indicus &lt;400&gt;

ggtggccaag cacgtgaaga atcctcctgc aatgtgtgag acctgcacac aatgtgtttg 60 agccttgggt tgagaagatc ctctggagaa gggaaaggct atctattcca gtattctggc 120 ctgaagaatt acaaattcca tacactgtat gatccatggg attgcaaaaa gtcagacatg 180 actaagcaac tttcacttac ccagagagat acaaaataag cattgatgaa agaaagcaaa 240 acacatatag atggagaaac ataccatgtt cttagattgg attaattgat atagttaaaa 300 tgagtatact acccaaaaca atcaatagat tcaatgcaat acctacaata tttttcataa 360 aactagaaca aattatttta tgatttgcat ggaaacacat aagactttga agagacaaag 420 caattttgag aaagaagaat gaaactagaa gagtcaaact ttttgataag actacaccac 480 aaagatatca agagagtatg aaacttgcac aaacatagaa atacagacca atggaacaaa 540 atataaagtc caaagataaa ttcatgcacc cattatcttt gacaatggag gcaaaaatat 600 gcaaccaaaa aatatatttt cttcaaaaag tgttactggg aaaactggac agctatgtgt 660 agaaactaga acagtatcta acatcaagtt caaaataaac tcaaaatgga tttacagatg 720 taaatgtaag accagaaaat ataaaaccct tagagtaaaa cataggtata accactctaa 780 aataagacac agcaagatcc tctatgaccc attgcctaga gtaaaggaaa taaaaacaaa 840 attaaaaaaa aaaagcaacc taattaagct ctatagcttg tgtacaatga ggaaactata 900 attaaggtga atatgtagcc ttcagtttta tgtgattatg gtttcagtgt atctgtcctc 960 tgatgccctc ttgcaacacc taccatcttt tttggatttc tcttaccttg gtcatggggt 1020 atctcttcac ggctgctcca gcaaagcgca gccattgctc cttaccttgg atgaggggta 1080 tctcctcacg gcacctatcc tgaaattgaa catggaatag ctcctctagg ccctcctgag 1140 cccatgcagc caccgctcct tggatctggg gttactcatc tcagctgctg ccccaacttt 1200 gggtgtgggg tagctgctct tggctgctct tgtgctgtcg cagccttgca ctgtccgcca 1260 cagcccctga cttctgacac ggtttagctc ctccctgctg tgcccctgac ctttgacacg 1320 2 1325058 ggtaagcttc tctcggctac agcccctgac ctcggacgtg gggtagctcc tcccagccat 1380 cgcccctgac ctcagacgtg gggtagctcc tatgggctgc tccagcgctg ttgtagcctt 1440 gctctctcag ctgctggccc tgacctcgga catggggtag ctcctctggg ctgctcctgc 1500 gctgttgcag ccttggaatc ttggccacc 1529 &lt;210&gt; 7 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; Bos indicus &lt;400&gt; 7 24 tcctcctgca atgtgtgaga cctgggtggccaag cacgtgaaga atcctcctgc aatgtgtgag acctgcacac aatgtgtttg 60 agccttgggt tgagaagatc ctctggagaa gggaaaggct atctattcca gtattctggc 120 ctgaagaatt acaaattcca tacactgtat gatccatggg attgcaaaaa gtcagacatg 180 actaagcaac tttcacttac ccagagagat acaaaataag cattgatgaa agaaagcaaa 240 acacatatag atggagaaac ataccatgtt cttagattgg attaattgat atagttaaaa 300 tgagtatact acccaaaaca atcaatagat tcaatgcaat acctacaata tttttcataa 360 aactagaaca aattatttta tgatttgcat ggaaacacat aagactttga agagacaaag 420 caattttgag aaagaagaat gaaactagaa gagtcaaact ttttgataag actacaccac 480 aaagatatca agagagtatg aaacttgcac aaacatagaa atacagacca atggaacaaa 540 atataaagtc caaagataaa ttcatgcacc cattatcttt gacaatggag gcaaaaatat 600 gcaaccaaaa aatatatttt cttcaaaaag tgttactggg aaaactggac agctatgtgt 660 agaaactaga acagtatcta acatcaagtt caaaataaac tcaaaatgga tttacagatg 720 taaatgtaag accagaaaat ataaaaccct tagagtaaaa cataggtata accactctaa 780 aataagacac agcaagatcc tctatgaccc attgcctaga gtaaaggaaa taaaaacaaa 840 attaaaaaaa aaaagcaacc taattaagct ctatagcttg tgtacaatga ggaaactata 900 attaaggtga atatgtagcc ttcagtttta tgtgattatg gtttcagtgt atctgtcctc 960 tgatgccctc ttgcaacacc taccatcttt tttggatttc tcttaccttg gtcatggggt 1020 atctcttcac ggctgctcca gcaaagcgca gccattgctc cttaccttgg atgaggggta 1080 tctcctcacg gcacctatcc tgaaattgaa catggaatag ctcctctagg ccctcctgag 1140 cccatgcagc caccgctcct tggatctggg gttactcatc tcagctgctg ccccaacttt 1200 gggtgtgggg tagctgctct tggctgctct tgtgctgtcg cagccttgca ctgtccgcca 1260 cagcccctga cttctgacac ggtttagctc ctccctgctg tgcccctgac ctttgacacg 1320 2 1325058 ggtaagcttc tctcggctac agcccctgac ctcggacgtg gggtagctcc tcccagccat 1380 cgcccctgac ctcagacgtg gggtagctcc tatgggctgc tccagcgctg ttgtagcctt 1440 gctctctcag ctgctggccc tgacctcgga catggggtag ctcctctggg ctgctcctgc 1500 gctgttgcag ccttggaatc ttggccacc 1529 &lt; 210 &gt; 7 &lt; 211 &gt; 24 &lt; 212 &gt; DNA &lt; 213 &gt; Bos indicus &lt;400&gt; 7 24 tcctcctgca atgtgtgaga cctg

&lt;210&gt; 8 &lt;211&gt; 24 &lt;212〉DNA &lt;213〉Bos indicus &lt;400〉 8 24 caagattcca aggctgcaac agcg&lt;210&gt; 8 &lt;211&gt; 24 &lt;212>DNA &lt;213>Bos indicus &lt;400> 8 24 caagattcca aggctgcaac agcg

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Claims (1)

吏)正替換頁 年月.if 十、申請專利範圍： 1 . -種鑑別牛隻性別的方法’包含下列步驟 提供含有核酸之牛隻檢體； 提供一組或'组以上選自包含SEQ |D N _.5、及啦丨_7與_丨_8所組成= 核指引子對族群中’其中各組係包含兩條寡核皆酸引 子， 利用 SEQ ID NO. 4 及 SEQ ID N0_ 5、或 SEQ |D N〇 7及SEQ ID NO. 8的募核普酸引子對進行增幅反應；及 偵測增幅產物之存在與否，以決定該牛隻之性別其 中當增幅產物存在時所檢測之檢體性別為雄性。 2 ·如申睛專利範圍第1項所述之方法，其中增幅方 法為聚合酶連鎖反應試驗。 如申請專利範圍第1或2項所述之方法，其中該檢體 中所含核酸係同時與一内控制組引子對進行增幅。 4 ·如申請專利範圍第3項之方法，其中内控制組引 子對為 SEQ ID N0:1/SEQ ID N0:2。 5 ·如申請專利範圍第4項所述之方法，其中該檢體 為全血’血球、糞尿或***。 6 .如申請專利範圍第5項之方法，其中經SEQ ID N〇:4及SEQ ID NO:5寡核苷酸引子對所増幅之序列大小 約為315 b p。 7 .如申請專利範圍第5項之方法，其中經SEQ ID [S1 ] 1325058 ggrrr^a 年；.丨：:；s Ν〇··7及SEQ ID NO:8募核苷酸引子對所增幅之序列大小 約為 1500bp。 8 ·如申請專利範圍第6項之方法’其中經SEQ ID NO:4及SEQ ID NO:5寡核苷酸引子對所增幅之序列為 SEQ ID NO. 3 〇 9 .如申請專利範圍第6項之方法，其中經SEQ id NO: 7及SEQ ID NO: 8募核苷酸引子對所增幅之序列為seQ ID N〇.6。 1 ο. —種鑑別牛隻性別特異性序列之寡核苷酸引子 對’其係一組或一組以上選自包含SEQ id Ν〇· 4與SEQ ID N〇_ 5、及SEQ ID NO. 7與SEQ ID NO. 8所組成的的寡 核苷酸引子對族群中，其中各組係包含兩條寡核苷酸弓丨 子。 1 1 .如申請專利範圍第1 〇項所述之寡核苷酸引子 對’其中各組寡核苷酸引子對進而包括一内控制組寡核甘 酸引子對。 1 2 ·如申請專利範圍第1 1項所述之募核苷酸弓丨子 對，其中該内控制組寡核苷酸引子對為S E Q丨D Ν 0:1 /S E Q ID NO:2。 十一、圖式： 如次頁 2吏) is replacing the page year. The tenth, the scope of the patent application: 1. The method for identifying the sex of the cattle 'includes the following steps to provide a bovine sample containing nucleic acid; providing a group or a group above selected from the group consisting of SEQ | DN _.5, and 丨 _7 and _ 丨 _8 consist of = nuclear guide pair in the group 'where each group contains two oligo-acid primers, using SEQ ID NO. 4 and SEQ ID N0_ 5, Or the SEQ | DN 〇 7 and SEQ ID NO. 8 nucleotide acid primer pairs are subjected to an amplification reaction; and detecting the presence or absence of the amplification product to determine the sex of the cattle, wherein the detection is performed when the amplification product is present The body sex is male. 2. The method of claim 1, wherein the amplification method is a polymerase chain reaction test. The method of claim 1 or 2, wherein the nucleic acid contained in the sample is simultaneously increased with an internal control group primer pair. 4. The method of claim 3, wherein the internal control group primer pair is SEQ ID NO: 1 / SEQ ID NO: 2. 5. The method of claim 4, wherein the sample is whole blood 'blood, excrement or semen. 6. The method of claim 5, wherein the SEQ ID N: 4 and SEQ ID NO: 5 oligonucleotide primer pairs have a sequence size of about 315 b p. 7. The method of claim 5, wherein the nucleotide increase is increased by SEQ ID [S1] 1325058 ggrrr^a years; .丨::;s Ν〇··7 and SEQ ID NO:8 The sequence size is approximately 1500 bp. 8. The method of claim 6, wherein the sequence amplified by the primer pair of SEQ ID NO: 4 and SEQ ID NO: 5 is SEQ ID NO. 3 〇 9 as claimed in claim 6 The method of the present invention, wherein the amplified sequence of the nucleotide primer pair by SEQ id NO: 7 and SEQ ID NO: 8 is seQ ID N〇.6. 1 ο. an oligonucleotide primer pair that identifies a bovine gender-specific sequence 'a group of one or more selected from the group consisting of SEQ id 4·4 and SEQ ID N〇_ 5, and SEQ ID NO. 7 In the group of oligonucleotide primer pairs consisting of SEQ ID NO. 8, wherein each group comprises two oligonucleotides. An oligonucleotide primer pair as described in the first aspect of the patent application, wherein each group of oligonucleotide primer pairs further comprises an internal control group oligonucleotide primer pair. 1 2 . The nucleotide pairing pair described in claim 11 wherein the internal control group oligonucleotide primer pair is S E Q丨D Ν 0:1 /S E Q ID NO:2. XI. Schema: as the next page 2