在做選殖工作時,常需要限制酵素切割圖譜輔助實驗設計。若載體與要研究的基因的序列都被決定出來了,則可先用 StringSearch 由資料庫中找到序列,再用 Assemble 程式與 Rerverse 程式在電腦上剪接出新的序列。一旦有了序列就可利用「圖譜分析」下的 Map 程式,在核酸序列上顯示切割位置與蛋白質序列,或是用 MapPlot 繪出線狀的切割圖譜。若需知道切割後每個片段的大小可用 MapSort 程式,一旦有了 MapSort 程式的輸出檔,即可用 PlasmidMap 程式,繪出質體的圓形圖譜。這一系列的工作在學會使用 GCG 的技巧後應可自行操作,此外,在習題組解答中有操作步驟,可在陽明的 FTP 伺服器上取得。
為了確定搜尋出來的序列上之酵素切割位置,在 GCG 環境下使用下列指令啟動程式 map:
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/userdata/manager/project/e % map Map displays both strands of a DNA sequence with restriction sites shown above the sequence and possible protein translations shown below. (Linear) MAP of what sequence ? xxu17997 Begin (* 1 *) ?1 End (* 3344 *) ?3344 Select the enzymes: Type nothing or "*" to get all enzymes. Type "?" for help on which enzymes are available and how to select them. 說明:給定所欲搜尋的酵素名稱,大小寫不拘;或直接<return>則可得所有酵素在序列上之切割位置 Enzyme(* * *):EcoRI EcoRI "ECORI" selected 1 enzyme, new total: 1. Enzyme: BamHI BamHI "BAMHI" selected 1 enzyme, new total: 2. Enzyme: What protein translations do you want: a) frame 1 b) frame 2 c) frame 3 d) frame 4 e) frame 5 f) frame 6 t)hree forward frames s)ix frames o)pen frames only n)o protein translation q)uit Please select (capitalize for 3-letter) (* t *): n 說明:不必轉譯胺基酸序列,所以鍵入“n” What should I call the output file (* pGFP.map *) ? pGFP.map |
(show part of
pGFP.map here) B a m H I aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccggtagaaaaaatgag ...... 234 ------+---------+---------+---------+---------+---------+--- ttcgaacgtacggacgtccagctgagatctcctaggggcccatggccatcttttttactc E c o R I gaattccaactgagcgccggtcgctaccattaccaacttgtctggtgtcaaaaataatag ......1014 ------+---------+---------+---------+---------+---------+--- cttaaggttgactcgcggccagcgatggtaatggttgaacagaccacagtttttattatc |
以同樣方式,來找到 pgem7z 序列上的 EcoRI, BamHI 的切割位置分別在序列 43 與 78 上
在 GCG 的環境下,assemble 是一個剪接序列的程式,但需先自行設計剪接順序並計算片段大小及序列號碼,依序輸入,以剪接出所欲的新序列。
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/userdata/manager/project/e %assemble Assemble constructs new sequences from pieces of existing sequences. It concatenates the fragments you specify and writes them out as a new sequence file. SeqEd is a better tool for assembling sequences interactively, but Assemble is best for assembling sequences from fragments defined in a list file. ASSEMBLE from what sequence(s) ? pgem7zfp Begin (* 1 *) ? 1 End (* 3000 *) ? 43 Reverse (* No *) ? n That range begins ggcg and ends gagg. Is this correct (* Yes *) ?y 說明:輸入序列名稱及剪接片段的序列號碼,並檢查是否正確。 That is done, now would you like to: A)dd another segment from this sequence G)et segments from another sequence W)rite out this assembly into a file Please choose one (* W *): g ASSEMBLE from what sequence(s) ? xxu17997 Begin (* 1 *) ? 269 End (* ??? *) ? 1018 Reverse (* No *) ? y 說明:因為GFP基因的方向在pLT中與在pGFP中相反,所以必須將GFP的序列反過來。在使用reverse的功能時,務必注意使用另一股來計算切點之序列號碼,以得正確之接合點。 That range begins attc and ends cggg. Is this correct (* Yes *) ?y 說明:可直接利用此處所示的數個bases,檢查前後序列是否正確連接,或於剪接完成後,再利用程式map檢查限制酵素位置是否如預期。 That is done, now would you like to: A)dd another segment from this sequence |
G)et segments from
another sequence W)rite out this assembly into a file Please choose one (*W*):g ASSEMBLE from what sequence(s)?pgem7zfp Begin(*1*)?79 End(*3000*)?3000 Reverse(*No*)?n That range begins atcc and ends tata. Is this correct(*Yes*)?y That is done, now would you like to: A)dd another segment from this sequence G)et segments from another sequence W)rite out this assembly into a file Please choose one (* W *): w What should I call the output file (* pgem7zfp.seq *) ? pLT.seq 如此即完成圖3-1的序列剪接。 |
在GCG 環境下有另一電腦程式 reverse 可找到給定序列的反轉互補股。使用程式時,若在程式後加上檔名,程式即會自動執行。例如:
%reverse gfp-1.seq REVERSE-COMPLEMENT of: gfp-1.seq check: 2478 from: 1 to: 749 |
從輸出的檔案(gfp-1.rev) 內可看出序列已被改變為反方的互補股(粗體字部份):
FROMIG of:
/userdata/NYMU/uyang/liao/pGFP.seq locus xxu17997 3344 bp dna circular syn 28-jan-1995 definition cloning vector pgfp, complete sequence. accession u17997 keywords green fluorescence protein; gfp. source cloning vector pgfp. . . . gfp-1.rev Length: 749 September 1, 1995 11:20 Type: N Check: 5359 .. 1 aattctacga atgctatttg tatagttcat ccatgccatg tgtaatccca 51 gcagctgtta caaactcaag aaggaccatg tggtctctct tttcgttggg |
利用 mapplot 可繪製簡潔清楚的酵素切割相對位置之直線圖譜
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/userdata/manager/project/e %mapplot pLT.seq -cir -onc 說明:熟練之使用者可直接在指令後設定使用參數,以增加運算速度。 MapPlot displays restriction sites graphically. If you don't have a plotter, MapPlot can write a text file that approximates the graph. Process set to plot with VT340 attached to term using the regd graphic interface. Begin (* 1 *) ?1 End (* 3715 *) ?3715 Select the enzymes: Type nothing or "*" to get all enzymes. Type "?" for help on which enzymes are available and how to select them. Enzyme(* * *): When your VT340 attached to tty is ready, press <return>. 說明:須注意終端機或繪圖機之設定是否正確,再予以執行。 |
使用程式 plasmidmap 來產生質體圖譜
使用 plasmidmap 繪製質體之酵素圖譜時,需要兩個資料檔,其中 tick 檔儲存酵素的切割位置,ranges則儲存欲特別繪在質體圖上之標幟或序列範圍,其作業流程如圖3-2所示:
圖3-2畫質體圖譜的流程圖
使用 mapsort
酵素切割位置可直接利用 mapsort 程式自動產生在執行時必須加上〝-plasmid〞之參數才會產生正確的格式。
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/userdata/manager/project/e %mapsort -plasmid -once MapSort finds the coordinates of the restriction enzyme cuts in a DNA sequence and sorts the fragments of the resulting digest by size. MapSort can sort the fragments from single or multiple enzyme digests. (Circular) (Six-base) (Plasmid) MAPSORT of what sequence ? pLT.seq Begin (* 1 *) ?1 End (* 3715 *) ?3715 for help on which enzymes are available and how to select them. Select the enzymes: Type nothing or "*" to get all enzymes. Type "?" Enzyme(* * *): What should I call the output file (* pLT.tick *) ?pLT.tick Mapping ....... [ sun670 ] /userdata/manager/project/e % |
建立 ranges 檔
ranges 檔所存的是想繪在質體上之標幟,例如 Amp r (beta-lactamase)「基因」,插入的基因的名字 (GFP)等,ranges 檔須先經文字處理,建檔後才能送到 GCG 系統中執行。其中除了標出要寫的文字外,還需將其範圍序號寫出,若是循順時鐘的方向畫圖,則寫範圍時在〝From〞之下寫較小的數字,在〝To〞之下寫較大的數字,並在〝Strand〞下填入〝+ 〞。例如
Ranges for pLT.Seq:
assembled From genbank:pgem-7zpf id pgem7zfp preliminary; circular dna; syn; 3000 bp. and from pGFP cDNA vector Name From To Strand Color Symbol1 Symbol2 Style .. ! the genes from pGEM-7Zf(+) Beta_Lactamase(Amp_R) 2034 2894 + Black | | Block ! blocks for vector and insert GFP_coding_region 774 57 - Blue | |
若是逆時鐘方向繪圖則反之,如上示之 GFP,其範圍是先寫大的數字,再寫小的數字,在〝Strand〞下則填入〝-〞。在給定範圍的兩側可加〝<〞或〝|〞等符號,由使用者自行設計,最後在〝Style〞的部份下可指定〝Range〞或〝Block〞,前者會另繪一條線,後者則會緊貼著圓圈繪出一區間,其效果可在圖3-1上看到。
建立 fil 檔
一旦有了這兩個檔案後即可利用〝plasmidmap〞程式來繪圖。至於如果每一次繪不同的圖時都將檔案存為一樣的名字,例如 pLT.tick 與 pLT.ranges 那麼可將這兩個檔案名存在另一檔案中,其格式如下。要注意的是 tick 檔的檔名必須放在第一個。一旦有此檔案,每次在使用 plasmidmap 這程式時只要輸入此檔名即可。
Labelling files for
a PLASMIDMAP pLT.Seq .. pLT.tick pLT.ranges |
設定顯示圖譜於畫面上或由繪圖機繪製
Process set to plot
with VT340 attached to term using the regd graphic interface. |
系統詢問在哪個檔案中,存有要注意在檔名前加的"@",若忘記加就會產生錯誤訊息
PLASMIDMAP of what label file(s) ? @pLT.fil |
若在處理不同序列時,每次都會另給檔案名稱,則可用另一方式執行
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/userdata/NYMU/uyang/liao %plasmidmap -pass Process set to plot with HP7475 attached to -term using the hpd graphic interface. PLASMIDMAP of what label file(s) ? @plt.fil |
即可得到如圖3-1(c)之結果
Last updated on 08/30/01