Getting Started with DNA Dynamo

The following steps will introduce you to some of the basic features in DNA Dynamo..

1) Load a Genbank record from NCBI

(internet connection firewalls may have to be configured to allow JAVA to access the internet).

a) Enter the accession number U52953 (which is a genbank record for HIV-1 92BR025 from Brazil, complete genome) in the text box under the title 'GENBANK ACCESSION' found in the center of the DNA Dynamo main window - then press the adjacent 'GO'.

b) The DNA sequence for Genbank accession U52953 is placed in the sequence window - and a pop up window asks you which features you would like to import from the features defined in the Genbank record - for now, select the CDS (coding sequence) features by clicking the check box to the right of the CDS option - and press the 'Import Selected Features' button. Note that different combinations of features can be imported later by selecting menu options 'Files'->'Import'->'Import/Re-Import DNA and Features from Genbank Text in Notes'.

2) Examining the sequence you have imported.

a) Press the 'Notes' button on the main panel - you will see the text of the Genbank record - you may enter your own notes for any sequence- and even paste image data from the 'System Clipboard' into the notes section of every sequence window you use - image data can sometimes be useful for storing Vector Illustrastions for commercial vectors. Press the sequence button again to return to the DNA sequence.

It may be useful at this point to save this DNA Dynamo sequence. Select 'Save As' from the File menu, and enter a name (eg hivseq).

b) Press the 'Features' button to display the imported CDS features. The location of the various HIV-1 CDSs - such as gag, pol, env, rev and tat are illustrated. Note that the rev and tat CDSs are formed by splicing and that the exon/intron structure is illustrated. It is easy to add your own annotations to the DNA sequence, simply select the sequence to be annotated and press the 'Annotate' button. Features may also be added from the 'BLAST' results viewer. In addition to the graphic map, features can be drawn directly over the DNA sequence by pressing the "Draw Annotations' button. You can adjust the layout of annotations (text size, spacing etc using the menu options 'Sequence'->'Adjust Display Settings'. Annotations may also be displayed on multiple alignments of DNA/Protein significantly enhancing the readability of such displays. Annotated sequences and alignments can be printed to pdf, or saved as as an eps file, allowing import into dedicated graphics programs such as adoble illustrator, for further adjustment.

c) Place the mouse pointer over the 'env' CDS and click the mouse button once. A pop up menu presents several options - select 'Set as ORF' (ORF = Open Reading Frame) - note that a red forward arrow is placed on the DNA sequence before base 5583 and a black reverse arrow is placed on the DNA sequence after base 8153 - representing the start and stop translation points for the env gene.

d) Press the 'MODE' button positioned at the top left of the DNA sequence to switch to 'Translated Mode' - scroll to base 5583 and you will see the protein translation underneath the set open reading frame. ORFs can also be set by clicking on individual open reading frames in the 'ORF Map'

3) Editing and Saving Oligos in the oligo database.

a) with the mouse select the first 19 bp of the env ORF (ATGAGAGTGGAGGGGATAC) - and press the 'Oligo' button on the main panel - the oligo editor is opened

b) change the type of the oligo to PCR by clicking the radio button next to the PCR option.

c) place the cursor at the start of the oligo sequence and type in GGATCC to place a BamH1 site at the 5' end of the oligo - click the 'MODE' button and note that the translation start point is remembered - this is useful when designing oligos against an internal section of a coding sequence - also note that you can select an amino acid with the mouse and type in a new amino acid - you are presented with codon options for the new amino acid - useful for designing mutagenesis oligos.

d) enter a name for the oligo in the text box to the right of the 'Oligo Name' text (eg env 5' PCR with BamH1) and then select 'Save Oligo in Oligo Box' from the Oligo Editors file menu.

e) look at the features map again - you should see that a red oligo mark has appeared at the 5' end of the env CDS. Place the mouse pointer over the red oligo mark - click the mouse button and select 'PCR Oligo Forward' - the oligo will be drawn above its target sequence in the DNA sequence display and is set to be used as the forward oligo in a virtual PCR. Note that any DNA Dynamo window containing the target sequence for this new oligo will dislay an oligo hit in its features map, not just the DNA sequence window you created the oligo in.

f) create a second oligo against the 3' 18 nucleotides of env (GAAGCAGCTTTGCAATAA) as above except this time enter a XhoI site (CTCGAG) at the 3' end of the oligo and switch the direction of the oligo from sense to anti-sense by clicking the 'anti-sense' radio button with the mouse. Note the effect that clicking the anti-sense button has the the 'Required Oligo 5'->3'' display at the bottom of the window, which is the sequence of your required oligo in the 5' to 3' direction. Note that you can press the 'Copy to Clipboard' button and subsequently paste tha oligo sequence into another application such as an oligo order form on a web page etc).

g) The new 3' oligo appears on the features map at the 3' end of the env gene. Place the mouse pointer over the red oligo mark - click the mouse button and select 'PCR Oligo Reverse - the oligo will be drawn above its target sequence in the DNA sequence display and is set to be used as the reverse oligo in a virtual PCR.

4) Sub-cloning - you may join together insert dna sequences to vector sequences to help plan your subcloning experiments and draw vector maps.

a) select 'Subclone into Vector' from the 'Vector' menu on the main window displaying the HIV sequence - a 'Construct Maker' window opens.

b) select pBlueScriptSK+ from the sample vectors on the left hand side menu

c) press the 'Use PCR Product' button so that the top 'insert map' will represent the theoretical PCR product from the oligos you entered for the env CDS - you should see a BamH1 site indicated on the 5' end of the insert and a XhoI site indicated at the 3' end of the insert. (note - some users find it easier to simply edit the DNA sequence by adding the sites manually, rather than selecting oligo's like this)

d) with the mouse - press and drag the upper 5' BamH1 site of the insert to the lower BamH1 site of pBlueScript. a red line will join the two sites. Similarly join the XhoI site of the insert to the XhoI site in the vector

e) finally press the 'Create Clone' button - a new window is opened and the sequence represents the env CDS, PCRd with your two oligo and subcloned into the BamH1 and XhoI sites in pBlueScript.

f) press the 'features' button on the new constuct window to view the construct features. Note the pBlueScript sequencing oligos that appear on the map.

g) Select 'Circular Map' from the 'Vector' menu - the map title can be edited and moved with the mouse. - press the 'Add prototypes (unique)' followed by the 'Layout Enzymes' button to illustrate restriction sites. Note that enzyme names can be moved with the mouse. The size, position, rortation and colour of map features can be edited from the control panel.

-----------------------------------

DNA Dynamo contains many more functions. See the help pages for more advice, or feel free to use the 'Ask a Question' option in the File menu, or email support@bluetractorsoftware.co.uk directly to ask any questions you may have about DNA Dynamo functionality. We will get back to you as soon as we can.

Copyright © BlueTractorSoftware Ltd, all rights reserved