Getting Started with DNA Dynamo
1) Load a Genbank record from NCBI
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'.
(you can also copy a genbank record from the web, and paste it into the DNA
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 Illustrations 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 ‘All’ button to see a table of restriction sites, and the ‘sites’ button to
see a map of restriction sites. You can click enzymes in the table to add them to
the map. Click the ‘expand’ tab in the map to open the map in a separate window
you can also set a preference for this to always happen). Press the ‘Sequence’
button to return to the DNA sequence.
c) 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 adobe illustrator, for further adjustment.
d) 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.
e) 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'
f) You can press the ‘number’ check-box on the view control to number the DNA
and protein residues.
3) Editing and Saving Oligos in the oligo database.
DNADynamo can design cloning oligos for you (eg to add restriction sites, or for
seamless gibson cloning and infusion cloning) - but it is also worth learning first
how to create oligos manually
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
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. Note
that you might want to add additional 5’ bases to aid digestion.
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). You might want to add additional bases before the
xho site to aid digestion.
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
h) You could also design these oligos using the ‘Add Restriction Sites’ mode in the
4) Sub-cloning - you may join together insert dna
sequences to vector sequences to help plan your sub-
cloning 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
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, rotation 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
email@example.com 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
The following steps will introduce you to some of the basic features in DNA