VectorNTI使用介紹.ppt

1、Molecule Construction and Design Using Vector NTI 10 Advance,Yi-Bu Chen, Ph.D. Bioinformatics Specialist Norris Medical Library University of Southern California 323-442-3309 ,Workshop Outline,Overview of molecule types and creation methods Major steps and tools for molecule

2、 construction and design Construct a new DNA molecule Design a new DNA molecule Overview of Gateway and TOPO cloning Using simulated gel electrophoresis to analyze cloned products Useful online resources for molecule design purpose,Fundamental Molecule Types in Vector NTI,Basic Molecules (DNA/RNA/Pr

3、otein) Not built from component fragments Sequences and features are either entered by users or imported from other databases. Constructed DNA/RNA Molecules Built from one or more fragments (parent molecules, linkers, adapters, etc.) Automatically receive the Feature map and sequences from the paren

4、t molecules. Constructed Protein Molecules Translated from a coding sequence of a DNA molecule Does not receive the Feature map from its parent DNA molecule,Methods of Creating New Molecule in Vector NTI,Basic Molecules (DNA/RNA/Protein) Importing molecules or sequences Manually creating new molecul

5、es Constructed DNA/RNA Molecules Splicing exons of an intron-exon join feature of another molecules Constructing from compatible component fragments from other molecules Designing from components of a user-defined fragments list Back-translating from protein molecules from components of a user-defin

6、ed fragments list Constructed Protein Molecules Splicing exons of a gene Translated from DNA molecules,Molecule Construction vs. Molecule Design,Recipient and donor fragments are defined by users. Restriction sites are defined by users. When required, the methods of terminus modification are defined

7、 by users.,Recipient and donor fragments are selected by users. Restriction sites are analyzed and generated by Vector NTI. Methods of terminus modification are determined by Vector NTI.,Major Tools for Molecular Construction and Design,Fragment Wizard Construct fragments (define positions and termi

8、ni) Design recipient/donor fragments (define termini) Add defined fragments to the Goal Molecule Definition List The Goal Molecule Definition List Contains the list of fragments defined and added by the user using the Fragment Wizard Serves as the starting point of molecular construction and design

9、Designs from components of a user-defined fragments list The Construct/Design Molecule Dialog Box Allows users to set the construction parameters and design preferences Allows users to enter information about the new molecule,Major Steps for Molecule Construction,Use Fragment Wizard to define compon

10、ent fragments. Add defined fragments to the Goal Molecule List. Use the Construct Molecule Dialog Box to set construction parameters, including necessary terminus modifications. Name, select data and describe the new molecule. Verify and edit the component fragments in the Goal Molecule Definition L

11、ist. Initiate molecule construction.,A Molecule Construction Example,Task Clone a fragment from pBR322 into pUC19 Donor fragment: pBR322, 5EcoRI3AvaI Recipient fragment: pUC19, 5SmaI3EcoRI Getting started 1. Open pBR322 and pUC19 in Vector NTI 2. Arrange the display window to display 2 molecules on

12、the same screen: Select Menu Window Tile Vertical,A Molecule Construction ExampleStep 1: Describe component fragments in the Fragment Wizard,Define the recipient pUC fragment (5SmaI3EcoRI) 1. Activate the pUC Graphic Pane 2. Click Add Fragment to Goal List button to open the Fragment Wizard 3. 1st s

13、creen: select Construct fragment click Next 4. 2nd screen: Click on the SmaI (415 bp) site in the graphic pane to set the 5 terminus. Click Next 5. 3rd screen: hold the SHIFT key and click on the EcoRI site in the graphic pane to set the 3 terminus. Click Finish 6. Check the description of the fragm

14、ent in the New Fragment message box. Click Cancel to go back to the Fragment Wizard if there are errors, otherwise, click Add to List to add the 1st fragment to the Molecule Goal List. B. Define the donor pBR322 fragment (5EcoRI3AvaI) following the above steps,From the tool bar, click the Open Goal

15、List button Notice that the 1st fragment in the list is always considered as the “recipient” molecule; the order of the fragments can be changed by click the Up or Down buttons If no errors, click the Run button in the Lists screen,A Molecule Construction ExampleStep 2: Inspect the Goal List,A Molec

16、ule Construction ExampleStep 3: Describing the new molecule,In the Construct Molecule screen, enter name “Tutorial1” Click the Recipients Start button to define the start of the new molecule (can be set at any other positions) Click General Info button to enter more info in the General Data dialog b

17、ox (Description: Tutorial molecule#1; Extra-Chromosome Replication: Bacteria; Replicon Type: plasmid; Keyword: your last name) Click Add and then OK button to return to the Construct Molecule dialog box.,A Molecule Construction ExampleStep 4: Construct the new molecule/Initial attempt,Click the Cons

18、truct button, in the Insert Molecule to Subset dialog box, enter “Tutorial” as subset name, and then click OK button. Vector NTI warns incompatible fragments (recipient pUC19 fragments blunt 5 SmaI terminus cannot be matched with the pBR322 cohesive 3AvaI terminus). Click the OK button to acknowledg

19、e the messages and return to the Construct Molecule dialog box Click the Close button to return You now need to modify one of the donor termini to make it compatible with the recipient termini for successful ligation.,A Molecule Construction ExampleStep 4: Construct the new molecule/modify the termi

20、nus,To make the 2 fragments compatible, you need to modify the pBR322 cohesive 3 AvaI terminus (5overhang) into a blunt one. In the Lists screen, click to select the pBR322, then click the Edit button. In the Fragment of Molecule dialog box, click the Right Terminus button. In the Right Terminus dia

21、log Box, select Completely filled in in the Biochemical Operations section; then click OK.,A Molecule Construction ExampleStep 4: Construct the new molecule/complete the construction,After the terminus is modified, click the Run button on the Lists dialog box to launch the Construct Molecule dialog

22、box. Click the Construct button and select Tutorial as the subset, then click the Overwrite button. Close the emptied Lists dialog box and go to the window that displays the newly constructed Tutorial1 molecule. Inspect the new molecule and information in the Text Pane Component Fragments folder.,A

23、Molecule Construction ExampleStep 5: Re-construct the new molecule if needed,With the Tutorial Molecule #1 open in the Vector NTI, select Menu File Molecule Operations Advanced DNA/RNA Construct to open the Construct Molecule dialog box. Alternatively, in the Vector NTI Explorer window select the in

24、tended molecule right click the mouse choose Re-construct from the short-cut menu. Make any desired changes in the Construct Molecule dialog box, and click the Construct button to re-construct the molecule.,Major Steps for Molecule Design,1. Define the goal molecules Use the Fragment Wizard to defin

25、e recipient and donor fragments Place the fragments in the Goal Molecule Definition List in proper order. Inspect the Goal Molecule Definition List. Enter information for the new molecule in the Design Molecule dialog box. Set appropriate parameters and design preferences in the Design Parameter dia

26、log box. Start the designing process. Inspect the design plan under the Design Description folder in the Text Pane. If not satisfied, re-design the molecule by changing the goal molecule description or using different design parameters.,A Simple Molecule Design Example,Task Clone a fragment from pBR

27、322 into pUC19 Donor fragment: pBR322, the TC(R) signal Recipient fragment: pUC19, 5 500 bp, 3 250 bp Getting started 1. Open pBR322 and pUC19 in Vector NTI 2. Arrange the display window to display 2 molecules on the same screen: Select Menu Window Tile Vertical,A Molecule Design ExampleStep 1: Defi

28、ne the recipient and donor fragments,Define the recipient pUC fragment 1. Activate the pUC Graphic Pane and click the Add Fragment to Goal List button to open the Fragment Wizard 2. 1st screen: select Design Recipient fragment click Next 3. 2nd screen: for the 5 of the new fragment, select Set to a

29、position, and enter 500, click Next 4. 3rd screen: enter 250 in the Set to a Position box to define the 3 terminus, click Finish then Add to List button. Define the donor pBR322 fragment 1. Activate the pBR322 Graphic Pane and click the Add Fragment to Goal List button to open the Fragment Wizard 2.

30、 1st screen: select Design Donor fragment click Next 3. 2nd screen: Move the cursor to click/select the TC(R) signal. 4. Click Finish then Add to List button.,From the tool bar, click the Open Goal List button Notice the Design button is selected, confirming the Design Mode. Make sure the recipient

31、(pUC fragment) is listed first. Notice that the exact positions of donor are not defined yet (NODEF), but it must contain the TC(R) signal. If no errors, click the Run button in the Lists screen,A Molecule Design ExampleStep 2: Inspect the Goal List,A Molecule Design ExampleStep 3: Describing the ne

32、w molecule,In the Design Molecule screen, enter name “Tutorial2” Click the Recipients Start button to define the start of the new molecule as the start of recipient molecule. Click General Info button to enter more info in the General Data dialog box (Description: Tutorial molecule#1; Extra-Chromoso

33、me Replication: Bacteria; Replicon Type: plasmid; Keyword: your last name) Click Add and then OK button to return to the Design Molecule dialog box.,A Molecule Design ExampleStep 4: Prepare to design the new molecule,In the Design Molecule screen, click Design button Select the previously created Tu

34、torial subset for the new molecule, click OK to continue. In the Design Parameters dialog box, you can choose your restriction enzyme subsets, the transformation systems you use, and other parameters. In this example, select Palindromes/Non-Ambiguous REN subset, and leave other parameters at their d

35、efault value.,A Molecule Design ExampleStep 5: Configure preferences for molecule design,In the Design Parameter dialog box, click the Preference button Choose your preferred parameters to create new molecules. In this example: deselect Ligation-BluntBlunt option, so Vector NTI will ensure all fragm

36、ents have at least one cohesive end. Leave other parameters at their default setting. The Advanced Preferences allows you to change the way Vector NTI evaluates possible design paths. Click OK to accept all Preferences and return to the Design Parameters.,A Molecule Design ExampleStep 6: Design and

37、inspect the new molecule,After the design preferences are set, click the Start Design button. Close the emptied Lists dialog box and go to the window that displays the newly designed Tutorial2 molecule. Inspect the new molecule and info in the Text Pane.,Verify the restriction enzymes used in the de

38、sign process. Add them to the display by using the Molecular Display Setup dialog box Restriction Map Setup. Inspect Design Plan In the Text Pane, open the Design Description Folder and subfolder Step #1. Highlights of the bench instruction for creating the new molecule: No biochemical operations ne

39、eded to modify the termini as they are compatible. The selected cloning option gives the required orientation of the cloned pBR322 fragment in the pUC19 recipient One of the recipients restriction sites (SmaI) is lost after ligation, this gives a mean for pre-selecting properly ligated molecule befo

40、re transformation. The new restriction site (AfeI) in the recombinant molecule that does not exist in the recipient allows one to use restriction analysis of the clones. Vector NTI also recommends oligos or PCR primers for clone analysis. Vector NTI also lists restriction sites that can be used to i

41、solate the closed fragments. 3. Print the Design Description: Open the Design Description folder Step#1 subfolder, click the Print Active Pane button on the tool bar.,A Molecule Design ExampleStep 7: Inspect and print the design plan,A Molecule Design ExampleStep 8: Re-design the new molecule,With t

42、he Tutorial Molecule #2 open in the Vector NTI, select Menu File Molecule Operations Advanced DNA/RNA Design to open the Design Molecule dialog box, click Yes to overwrite the original task and start the new design. Alternatively, in the Vector NTI Explorer window select the intended molecule right

43、click the mouse choose Re-design from the short-cut menu. Make any desired changes in the Design Molecule dialog box, and click the Design button to re-design the molecule.,Advanced Molecule Design I Complicated Recipient,Task Insert SV40s LARGE_T gene from SV40 to the 2nd ApaLI site of BPV1. Donor

44、fragment: SV40 LARGE_T gene (no ApaLI site) Recipient fragment: BPV1 at 2nd ApaLI site; 5 ApaLI site must be retained ligation: no blunt-blunt Getting started 1. Open SV40 and BPV1 in Vector NTI 2. Arrange the display window to display the 2 molecules on the same screen: Select Menu Window Tile Vert

45、ical,Advanced Molecule Design with Complicated RecipientStep 1: Define the recipient and donor Fragments,A. Define the recipient BPV1 fragment 1. Activate the BPV1 Graphic Pane and click Add Fragment to Goal List button to open the Fragment Wizard 2. 1st screen: select Design Recipient fragment clic

46、k Next 3. 2nd screen: for the 5 of the new fragment, click on the label of ApaLI site #2 (7631) in the Graphic Pane, click Next 4. 3rd screen: select Save Site, and then click Next 5. 4th screen: to define the 3, press SHIFT+ Click on the same ApaLI site, Click Finish then Add to List button. B. Def

47、ine the donor SV40 fragment 1. Activate the SV40 Graphic Pane and click the Add Fragment to Goal List button to open the Fragment Wizard 2. 1st screen: select Design Donor fragment click Next 3. 2nd screen: Move the cursor to click/select the LARGE_T signal. 4. Click Finish then Add to List button.,

48、From the tool bar, click the Open Goal List button Make sure the recipient (BPV1 fragment) is listed first. Click on the SV40 fragment, and then click the Edit button to open the Fragment Editor dialog box Check the Inverted box to change the direction of the donor fragment to match the recipients d

49、irection and then click the OK button. (when the Inverted box is not checked, the system will design it either way).,Advanced Molecule Design with Complicated RecipientStep 2: Inspect the Goal Molecule Definition List,Advanced Molecule Design with Complicated Recipient Step 3: Describing the new mol

50、ecule,Click the Run button, in the Design Molecule screen, enter name “Tutorial3” Click the Recipients Start button to define the start of the new molecule as the start of recipient molecule. Click the General Info button to enter more info in the General Data dialog box (Description: Tutorial molec

51、ule#3; Extra-Chromosome Replication: Bacteria; Replicon Type: plasmid; Keyword: your last name) Click the Add and then OK button to return to the Design Molecule dialog box.,Advanced Molecule Design with Complicated Recipient Step 4: Prepare to design and set the preferences,In the Design Molecule d

52、ialog box, click the Design button Select the previously created Tutorial subset for the new molecule, click OK to continue. In the Design Parameters dialog box, leave all settings at their default values. Click the Preferences button, notice the blunt-blunt ligation box remains turned off. Leave ev

53、erything at their default settings click OK to accept the Design Preference return to the Design Parameters dialog box.,Advanced Molecule Design with Complicated Recipient Step 5: Design and inspect the new molecule,After the Design Preferences are set, click the Start Design button. Close the empti

54、ed Lists dialog box and go to the window that displays the newly designed Tutorial3 molecule. Inspect the new molecule and info in the Text Pane.,Verify the restriction enzymes used in the design process. Add them to the display by using the Molecular Display Setup dialog box Restriction Map Setup.

55、Inspect Design Plan In the Text Pane, open the Design Description Folder and subfolder Step #1. Highlights of the bench instruction for creating the new molecule: Recipient: partial digestion - 1 ApaLI site inside recipient fragment. Donor: both termini were cut and then filled in, and ApaLI linkers

56、 were attached to the blunt ends before full digestion. Ligation: cohesive termini at both junctions. Enzymes to analyze and isolate insert with correct orientation: AvrII and ApaLI. Vector NTI also recommends oligos or PCR primers for clone analysis. 3. Print the Design Description: Open the Design

57、 Description folder Step#1 subfolder, click the Print Active Pane button on the tool bar.,Advanced Molecule Design with Complicated Recipient Step 6: Inspect and print the design plan,Advanced Molecule Design II Complex Donor Fragment,Task Insert SV40s LARGE_T gene from SV40 to a pre-determined sect

58、ion of BPV1. Donor fragment: SV40 LARGE_T gene (5 end with 440 bp flank region, 3 end at NcoI site) Recipient fragment: BPV1 from #5000 to #2500 bp ligation: no blunt-blunt Getting started 1. Open SV40 and BPV1 in Vector NTI 2. Arrange the display window to display the 2 molecules on the same screen

59、: Select Menu Window Tile Vertical,Advanced Molecule Design with Complex DonorStep 1: Define the recipient fragment,Activate the BPV1 Graphic Pane and click the Add Fragment to Goal List button to open the Fragment Wizard 1st screen: select Design Recipient fragment click Next 2nd screen: select Set to a Position and enter 5000 as the start for the 5 of the new fragment, click Next 3rd screen: select Set to a Position and enter 2500 as the start position of 3 for the new fragment, then click Finish then Add to List button.,Advanced Molecule Design w