Getting Started with DNA Dynamo
1) Load a Genbank record from NCBI
a) Use the ‘Download’ menus -> Retrieve a Genbank Accession Number’ (or Use ‘View’ -> ‘Show Info Panel‘ as shown in the graphic) then enter the
accession number U52953 (which is a genbank record for HIV-1 92BR025 from Brazil, complete genome) . You can also copy a genbank record from the
web, and paste it into the DNA window.
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 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. 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 PCR.
h) You could also design these oligos using the ‘Add Restriction Sites’ mode in the ‘Construct Maker’
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 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, 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 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
If you haven’t already downloaded DNADynamo the best way to get started and learn how to use the software is to download it and use it.
This video will introduce you to some of the basic functions in DNADynamo. You can also read a text based introduction
below, which covers some additional options. We’re hoping to update these help pages soon.
DNADynamo
BlueTractorSoftware
•
Windows
•
OSX
•
Linux
Getting Started with DNA Dynamo
1) Load a Genbank record from NCBI
a) Use the ‘Download’ menus -> Retrieve a Genbank Accession Number’ (or Use ‘View’ -> ‘Show Info Panel‘ as
shown in the graphic) then enter the accession number U52953 (which is a genbank record for HIV-1 92BR025
from Brazil, complete genome) . You can also copy a genbank record from the web, and paste it into the DNA
window.
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 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. 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 PCR.
h) You could also design these oligos using the ‘Add Restriction Sites’ mode in the ‘Construct Maker’
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 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, 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 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
If you haven’t already downloaded DNADynamo the best way to get started and learn how to use the software is to download it
and use it.
This video will introduce you to some of the basic functions in DNADynamo. You can also read a text based
introduction below, which covers some additional options. We’re hoping to update these help pages soon.
Getting Started with DNA Dynamo
1) Load a Genbank record from NCBI
a) Use the ‘Download’ menus -> Retrieve a Genbank Accession Number’
(or Use ‘View’ -> ‘Show Info Panel‘ as shown in the graphic) then enter the
accession number U52953 (which is a genbank record for HIV-1 92BR025
from Brazil, complete genome) . You can also copy a genbank record from
the web, and paste it into the DNA window.
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
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. 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
PCR.
h) You could also design these oligos using the ‘Add Restriction Sites’ mode in the
‘Construct Maker’
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
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, 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
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
If you haven’t already downloaded DNADynamo the best way to get started and learn
how to use the software is to download it and use it.
This video will introduce you to some of the basic functions in
DNADynamo. You can also read a text based introduction below,
which covers some additional options. We’re hoping to update
these help pages soon.
Alternatively you can copy and replace fragments from a sequence window
Getting Started with DNA Dynamo
1) Load a Genbank record from NCBI
a) Use the ‘Download’ menus -> Retrieve a Genbank
Accession Number’ (or Use ‘View’ -> ‘Show Info
Panel‘ as shown in the graphic) then enter the
accession number U52953 (which is a genbank
record for HIV-1 92BR025 from Brazil, complete
genome) . You can also copy a genbank record from
the web, and paste it into the DNA window.
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 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.
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 PCR.
h) You could also design these oligos using the ‘Add
Restriction Sites’ mode in the ‘Construct Maker’
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 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, 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
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
If you haven’t already downloaded
DNADynamo the best way to get started and
learn how to use the software is to download it
and use it.
