BIOS816/VBMS818 Lecture 4 - Restriction mapping

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BIOS816/VBMS818 Lecture 4 - Restriction mapping
Molecule Construction

• Guoqing Lu
• Office E115 Beadle CenterTel (402)
  472-4982Email glu3_at_unl.eduWebsite
  http//biocore.unl.edu

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Introduction - Restriction Mapping

• A description of restriction endonuclease
  cleavage sites within a piece of DNA
• Restriction-recognition sites
• short DNA sequences recognized and cleaved by
  various restriction endonucleases

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Introduction - Molecule Construction
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Programs Available for Restriction Mapping

• GCG
• map
• mapsort
• mapplot
• Vector NTI
• EMBOSS
• cirdna
• restrict

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GCG Command Line

• Logon to biocomp2 account
• map
• gb_sysynpuc18cv
• region from 200 to 500
• ECORI, BAMHI
• Six frames
• mapsort circular
• gb_sysynpuc18v
• whole region
• alui

Demo
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GCG - seqlab

• Set up the environment which allows seqlab
  running

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seqlab - map
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Vector NTI

• Suite of programs
• Fully integrated
• Sequence files/analysis/data stored in a separate
  database
• Layout
• Three (or more) panes, all of which are linked
• Sequence, text, graphics
• Toolbars, menus

DEMO
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Launching VNTI
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Vector NTI Databases

• Contain molecule and subbase information
• Eight different types of databases

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Database Maintenance

• From the Database menu
• Database Backup (perform at least weekly)
• Database Restore (if your hard drive crashes)
• Database Cleanup (to clean up the database
  suggested frequency weekly to monthly)

• NOTE no single file on a PC hard drive that
  contains all the information for any one molecule

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Opening and Viewing Molecules

• From the Vector NTI Explorer double-click on the
  molecule pBR322 in the DNA/RNA database
• The Molecule Viewer has a three-pane format/view
  - each pane is integrated to reflect each others
  actions
• Each pane can be activated by clicking within it
  or clicking on the appropriate icon at the
  left-hand side of the lower toolbar

Note lower toolbar is context sensitive
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Text Pane

• Contains molecule information
• e.g. Author, Comments
• stored in hierarchical folders
• The Feature Map folder
• Information on molecule features standardized to
  the GenBank format

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Graphics Pane

• Automatically generated from the data in the
  molecule file
• The arrow directions indicate which strand
  (direct or complementary) a feature (e.g. CDS) is
  coding by
• Hover the mouse cursor over a feature to view a
  tip window describing the feature

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Changing in the Graphics Pane

• Right-click on any CDS (orange arrow)
• choose CDS Display Setup
• Show Symbol field, click More
• Change the color to blue and click OK
• Click OK again
• All features of this type (CDS) will be displayed
  according to the new settings
• Choose View Display Profile Save Settings As
• Type Blue and click OK (click Yes if asked to
  save unused styles)
• Choose View Display Profile (Default)

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Sequence Pane

• Displays the primary sequence of the open
  molecule
• Restriction sites, motifs and translations may
  also be indicated here

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Changing Sequence Pane Display Settings

• Right-click in Sequence pane and choose Display
  Setup then click Sequence Setup
• Change the Length of Block to 20, Blocks per line
  to 5 and change the font style and color
• Click OK then OK again

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Restriction Map Setup

• Choose View Display Setup -gt Click RMap Setup
• Choose Analyses Restriction Analyses
  Restriction Fragments
• Restriction Map folder in the Text pane
• reflects those enzymes defined
• restriction enzyme sites

Do it yourself
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Save Restriction Results

• Highlight the contents of Restriction Map folder
• Click the Camera icon, check Selection and
  Clipboard then click Copy
• Double-click on the Comments folder, paste in the
  restriction fragments analysis and click OK
• Choose Molecule Save As and save to the
  database, overwriting the existing molecule

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Generating a Report of All Restriction Enzymes

• Choose Analyses Restriction Analyses
  Restriction Report
• Click Save, name the report and save the file to
  your desktop

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Construction Mode of CloningExample 1

• Create a new molecule by cloning the GAG from
  pNL4-3 into pGEX-5G/LIC, using the EcoRI
  restriction sites.

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Defining the Cloning Vector

• Open pGEX-5G/LIC
• Click on the EcoRI site at 961bp
• Shift-click on this site again to select EcoRI as
  the sole cloning site
• Choose List Add Fragment to Goal List and check
  Construction fragment (the first option)
• Click Next to view a summary of the 5 terminus
  then click Next again to view details of the 3
  terminus
• Click Finish then click Add to List
• Close the Molecule Viewer for pGEX -5G/LIC

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Define the Insert

• Open pNL4-3 from the Vector NTI Explorer
• Highlight the GAG CDS (bp 790-2292)
• Choose Analyses Primer Design Find PCR
  primers
• Change the minimum product length to 1503 (i.e.
  the same value as the maximum product size)
• Click the More button at the bottom right of the
  dialog box and remove any existing user-defined
  primers
• Click the button (by Attach to 5 terminus of
  Sense Primer) and select EcoRI from the list of
  enzymes
• Repeat for the Antisense primer then click OK
• Select the first product (1515bp in length)
• Right-click on the name, choose Save to Database
  and Create Window and save to the database as
  GAG PCR Product

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Molecule Construction

• With GAG PCR Product opened, Select the 5' and
  3' EcoRI sites at 2 and 1511bp respectively
  (click on the 5 site, shift-click on the 3
  site)
• Choose List Add Fragment to Goal List (select
  Construction fragment)
• Review the 5 and 3 termini by clicking through
  the Nexts then click Finish then Add to List
• Choose List Molecule Goal List to see a summary
  of the 2 fragments
• Click Run -gt Name the molecule pGEX-GAG
• Check Recipients Start for Start Position
• Click Construct, select a subbase then click OK
• Close the Lists dialog box

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Check Component Fragments

• Open the Component Fragments folder in the Text
  pane
• Optional Click on the hyperlink (in blue and
  underlined) to pGEX -5G/LIC or the GAG PCR
  product to open the parent molecule
• Select pGEX-5G/LIC in the Component Fragments
  folder
• Right-click and choose Find Component Fragment to
  highlight the sequence that originated from the
  vector

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Construction Mode of Cloning Example 2

• Dealing with incompatible termini, applying
  biochemical operations
• Open pUC19 from the Vector NTI Explorer
• Select 5 terminus BamHI 3 terminus SmaI
• Choose List Add Fragment to Goal List
• Click Finish then Add to List
• open YOL056W
• Make the selection 5 terminus PstI and 3
  terminus BamHI
• Choose List Add Fragment to Goal List
• Click Finish then Add to List
• Choose List Molecule Goal List
• Click the Run button to open the Construction
  Window
• Name the molecule pUC-YOL
• Click Construct
• Select the MAIN DNA/RNA database then click OK
• Click OK, then OK again at second warning message
• Click Close in the Construction Window
• Close the Lists window

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Dealing with incompatible termini

• Go to the Vector NTI Explorer, highlight pUC-YOL,
  right-click and choose Re-Construct
• Highlight the YOL056W fragment in lower box
• Click Edit, then click Left terminus (PstI site)
• Under Biochemical operations, choose S1-treated
  from the drop-down menu
• Click OK, OK again then Construct
• Save the construct to the MAIN database and
  overwrite existing version of pUC-YOL
• Examine the Component Fragments folder in the
  Text pane
• Close the Molecule Viewer

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Exercise

• Select a Recipient fragment
• pUC19
• Define what we want to keep
• Select a Donor fragment
• pBR322
• Define what we want to clone
• Clone pBR322 fragment into pUC19
• Do restriction analysis to confirm successful
  cloning