標準分析 - 電腦上的基因剪接

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。

尋找限制酵素切割位置

尋找出 BamHIEcoRI 的切割位置

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:

[ sun670 ] /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 78

序列的剪接

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。

[ sun670 ] /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

 

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

 

圖譜的繪製

Last updated on 08/30/01