三、 網頁實作
5.3 未來工作
目前MOI 在鑑定物種大都是依據 16s RNA 與 tRNA 兩種 structural RNA，未來可
能會加入housekeeping gene，以及提供使用者自行加入註解，希望能提供使用者
更多引子對的選擇。在最後模擬電泳圖的結果呈現上，希望能準確的預測出當
TM 變化時，模擬電泳圖也隨著 TM 變化而造成產物形狀、顏色也會隨之不同。
。
16
六、結論
在這個研究中，我們利用了在不同物種之間，它們的structural RNA 間距離不
同作為一個genomic feature，讓使用者可以利用的網頁介面，將想要鑑別的微生物群
組上傳到系統網頁，系統會依照使用者的需求設計最佳引子對組，進而利用這些詳
細的引子資訊去鑑別出使用者感興趣的微生物，而使用者可以得到詳細的引子資訊，
與聚合酶連鎖反應產物詳細資料，且會呈現模擬電泳圖供使用者參考。
17
Organism Sn 1 2 3 4 5 6 7 8 9
Bacillus subtilis subsp. subtilis str. 168 1 X 7 10 4 7 7 5 9 6
Bacillus anthracis str. Ames 2 X X 7 5 7 8 8 6 9
Bacillus clausii KSM-K16 3 X X X 5 7 9 9 7 9
Lactobacillus reuteri JCM 1112 4 X X X X 3 4 5 6 5 Lactobacillus fermentum IFO 3956 5 X X X X X 7 8 7 8 Bacillus subtilis subsp. spizizenii str. W23 6 X X X X X X 3 9 3 Bacillus atrophaeus 1942 7 X X X X X X X 8 4 Bacillus cellulosilyticus DSM 2522 8 X X X X X X X X 9 Bacillus subtilis subsp. spizizenii TU-B-10 9 X X X X X X X X X 表 1 引子對索引表
18
圖 1 Genomic feature
圖中三條Genome 都具有 ValtRNA 與 CystRNA，故利用 ValtRNA 與 CystRNA 間
的距離當作一項Genomic feature，當這段距離在 A、B、C 三條不同 Genome 時，
若彼此之間會有明顯的不同，就可以此來當作鑑別Genome 的依據，這邊限制的
差距必須在 50bp 以上，才會列入考慮。
19
圖 2 系統流程圖
20 Structural
RNA 註解
tRNAscan-SE NCBI 16SMicrobial
Database BLASTn
圖 3 structural RNA 的註解
Structural RNA 的註解分為兩部分(1)16S rRNA 部分利用 Blastn 比對 NCBI 的 16S
Microbial Database (2)其餘 tRNA 利用 tRNAscan-SE 此工具作註解。
21
Structural RNA Genome
3500
1500 50
圖 4 篩選 structural RNA pair
聚合酶反應的產物通常都在 100bp 到 3000bp 之間，過長容易不穩定，過短
則不易觀察，因此在這步驟裡會逐步篩選 structural RNA pair，若在範圍內的才會
進入下一步篩選。
22
我們設定兩個structural RNA pair 間的距離至少需要超過 50bp 才有具有鑑別
力。
23 rRNA sequence
Multiple sequences alignment [ClustalW]
Conserved region Database
PrimerHunter
Primer
圖 6 引子設計流程(1)
當我們得到structural RNA 的註解時，我們會將得到這些 rRNA sequence 去執
行ClustalW，找到一或多組的 conserved region，而這些 conserved region 會先存入資
料庫，讓PrimerHunter 做為設計引子的候選。
24
ValtRNA Ile tRNA ValtRNA CystRNA
ValtRNA Ile tRNA ValtRNA CystRNA
ValtRNA
CystRNA
Genome A Genome C
Genome D Genome B
Design common primer
Design specific primer
圖 7 引子設計流程(2)
若在不同的基因體上，某組structural RNA 具 highly conserved 現象，在大多數
的基因體上都有重複，我們會將它設計為common primer，若否，則另外設計 specific primer，如此可以避免設計重複的引子，節省大量的時間。
25 E-PCR Structural RNA
Primer
Genome A
Genome A
?
Tm值計算
圖 8 判斷是否黏合
得到引子之後，雖然引子都是針對 structural RNA 量身定做的，但由於不確
定設計出的來引子會不會黏到基因體上其他位置，所以我們利用E-PCR 預測出可
能接合的位置，再利用Tm 值計算公式去判斷是否會接合。三角形代表實際可以
黏合位置。
26
Geonme A Geonme B Geonme C Geonme D Geonme E
Geonme A 6 2 6 5
27
Geonme A Geonme B Geonme C Geonme D Geonme E
Geonme A ● ○ ● ○
28
圖 11 系統首頁
在此選擇要使用乳酸菌範例或者自訂上傳序列。
29
圖 12 乳酸菌範例
選擇乳酸菌菌株與設定參數，可設定溫度差與產物長度差異的門檻限制。
30
圖 13 使用者上傳頁面
使用者可自行上傳欲鑑定的基因體序列，可設定溫度差與產物長度差異的門
檻限制，並需要填入信箱與專案名稱，當完成分析時會傳送信件通知使用者。
31
圖 14 結果圖
Primer pair combination 28 代表第 28 組引子對解，此組結果用 6 個引子即可鑑定。
PCR reaction 為網頁超連結，點擊觀看引子對的詳細資料。
32
圖 15 引子對解(1)
Ｖ代表可以鑑別，ｘ則代表否。
圖 16 預測電泳圖(1)
圖中黃線代表電泳的結果，右側則為marker。
33
圖 17 引子對解(2)
圖 18 預測電泳圖(2)
34
圖 19 引子對解(3)
圖 20 預測電泳圖(3)
35
Temperature = 45
Temperature = 50 Temperature = 55
Result
圖 21 預測電泳圖(4)
黃色的線條代表預測會出現的產物，綠色則代表不是預期在這組 TM 會出現的產
物。聚合酶連鎖反應的產物會隨著 TM 的上升而有所改變。
36
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