U.S. patent application number 15/259033 was filed with the patent office on 2017-03-09 for methods and systems for in silico experimental designing and performing a biological workflow.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Kevin Clancy, Kok Hien Gan, Maurice LING, Kin Chong Sam, Raymond Tecotzky.
Application Number | 20170068431 15/259033 |
Document ID | / |
Family ID | 47561840 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170068431 |
Kind Code |
A1 |
LING; Maurice ; et
al. |
March 9, 2017 |
METHODS AND SYSTEMS FOR IN SILICO EXPERIMENTAL DESIGNING AND
PERFORMING A BIOLOGICAL WORKFLOW
Abstract
Embodiments describe a non-transitory computer-readable storage
medium encoded with instructions, executable by a processor,
comprising instructions for a method for performing a biological
workflow in silico comprising: presenting to a user a plurality of
subroutines listed in a sequential order of the workflow, wherein
at least two subroutines comprise two steps; providing the user
ability to navigate to any subroutine and/or step, to select a
subroutine, to view, set, or change one or more parameters
associated with step/subroutine; providing option to display
biomolecule(s) resulting from execution of the subroutines/steps;
and providing an option to navigate to a prior subroutine and
change a parameter of a step of the prior subroutine and execute
the step of the prior subroutine, if the user is not satisfied with
the displayed biomolecule(s). Computer systems and methods for
performing a biological workflow in silico are also described.
Inventors: |
LING; Maurice; (Singapore,
SG) ; Gan; Kok Hien; (Johor Bahru, MY) ;
Clancy; Kevin; (Carlsbad, CA) ; Tecotzky;
Raymond; (Carlsbad, CA) ; Sam; Kin Chong;
(Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
47561840 |
Appl. No.: |
15/259033 |
Filed: |
September 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13724765 |
Dec 21, 2012 |
9465519 |
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15259033 |
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61578820 |
Dec 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06F 3/0484 20130101;
G06F 3/04847 20130101; G16B 50/00 20190201; G16B 45/00 20190201;
G06F 3/0482 20130101 |
International
Class: |
G06F 3/0484 20060101
G06F003/0484; G06F 19/26 20060101 G06F019/26; G06F 3/0482 20060101
G06F003/0482 |
Claims
1. A non-transitory computer-readable storage medium encoded with
instructions, executable by a processor, the instructions
comprising instructions for providing a method for performing a
biological workflow in silico comprising: presenting to a user a
plurality of subroutines that comprise the biological workflow
listed in a sequential order, wherein at least two subroutines of
the plurality of subroutines comprise two steps; providing the user
ability to navigate to any subroutine of the plurality of
sequential subroutines, to select a subroutine and view, set, or
change one or more parameters associated with a step of the
selected subroutine; providing an option to display to the user,
one or more biomolecules resulting from execution of one or more of
the plurality of subroutines; and providing an option to the user
to navigate to a prior subroutine and change a parameter of a step
of the prior subroutine, if the user is not satisfied with the one
or more displayed biomolecules.
2-7. (canceled)
8. A non-transitory computer-readable storage medium of claim 1,
the method further comprising: providing the user the ability to
navigate to any subroutine of the plurality of sequential
subroutines to select a subroutine and view, set, or change one or
more parameters associated with a step of the selected subroutine,
wherein every parameter in a workflow that is set based on
ambiguous user data is highlighted to a user such that the
parameter can be viewed, set, or changed by the user.
9. The non-transitory computer-readable storage medium of claim 1,
wherein the instructions further comprise instructions for:
providing to the user the ability to save a log of all selected
parameters for each step of the user defined method; and receiving
and storing from the user at least one comment about the user
defined method.
10. The non-transitory computer-readable storage medium of claim 1,
wherein the instructions further comprise instructions for: storing
one or more external files uploaded by the user; and providing to
the user or to additional users ability to view the one or more
external files uploaded.
11. (canceled)
12. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
the one or more parameters viewed, selected, set or changed by the
user are from one or more default parameters, wherein default
parameters are pre-determined parameters stored in the
computer-readable storage medium.
13. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
the one or more parameters viewed, selected, set or changed by the
user comprise at least one user input parameter.
14. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 13, wherein
the user input parameter is a modified default parameter, a
parameter input by user, or a parameter imported by the user into
the computer system, wherein a default parameter is a
pre-determined parameter stored in a component of the
computer-readable storage medium.
15. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
the one or more parameters viewed, selected, set or changed by the
user comprise a combination of one or more default parameters and
one or more user defined parameters, wherein default parameters are
pre-determined parameters stored in a component of the
computer-readable storage medium and wherein the user input
parameter is a modified default parameter, a parameter input by
user, or a parameter imported by the user into the computer
system.
16. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
providing the user ability to navigate to any subroutine of the
plurality of subroutines is by a graphical user interface (GUI)
which comprises displaying on a first display screen pane all the
subroutines of a sequential subroutine comprising the biological
workflow and, following selection by the user of any one
subroutine, displaying on a second display screen, one or more
steps associated with the selected subroutine.
17. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
providing the user the ability to navigate to any step of a
subroutine is accomplished by a graphical user interface (GUI)
which comprises displaying on a first display screen pane the
subroutine and displaying on a second display screen, one or more
steps associated with the selected subroutine.
18. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
providing to the user an option for saving on the computer readable
storage medium a user defined method comprising at least one
selection desired by the user comprises: receiving selections by
the user of one or more parameters for each step of the plurality
of subroutines of the biological workflow and saving the selections
as a selected plurality of steps, wherein the saved selected
plurality of steps comprise the user-defined method; and storing a
user defined name for the user-defined method.
19. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
providing to the user an option for saving on the computer readable
storage medium a user defined method comprising at least one
selection desired by the user comprises: a) displaying on a display
screen pane a prescribed plurality of subroutines in a sequential
order of the biological workflow, wherein the prescribed plurality
of subroutines are comprised in a computer readable format; b)
navigation by a user using a graphical user interface (GUI) on the
display screen of each subroutine of the prescribed plurality of
subroutines of the biological workflow; c) selection by the user of
one of the subroutines; d) navigation by a user using a GUI on the
display screen of each step of a selected subroutine; e) selection
by the user of one or more parameters in each step of a selected
subroutine to obtain a modified plurality of steps; f) storing the
modified plurality of steps by the user, wherein the stored
modified plurality of steps comprise the user defined subroutine g)
repeating steps b)-f) till all the plurality of sub routines are
stored as user defined subroutines; and h) saving and executing the
plurality of user defined subroutines to perform a user defined
biological workflow.
20. (canceled)
21. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, of claim 1, wherein
the navigation by a user of any subroutine or step of the workflow
is in a non-sequential order of the steps or the subroutine.
22. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor of claim 15, further
comprising: executing the user defined workflow comprising
executing in silico all the steps of the user defined workflow in
sequential order; viewing a first biomolecule obtained by executing
the user defined workflow in silico; generation of at least a
second user defined workflow, comprising changing the selection of
at least one parameter to have a different value relative to the
same parameter that was selected in claim 1; executing in silico
the at least second user defined workflow to obtain a second
biomolecule; viewing the second biotechnology product in silico;
and comparing the first biomolecule with the second biomolecule,
thereby allowing a user to determine if the first user defined
workflow or the second user defined method produces a preferred
biomolecule.
23. A non-transitory computer-readable storage medium encoded with
instructions, executable by a processor, of claim 1, wherein the
biological workflow comprises a cloning method, a recombination
method, a ligation method, a vector designing method, a method for
synthesis of a nucleic acid, a primer design method, a method for
synthesis of a polypeptide, method for analysis of a cloned
molecule, method of protein analysis, method for making a modified
host.
24. A non-transitory computer-readable storage medium encoded with
instructions, executable by a processor, of claim 1, the
instructions comprising instructions for: providing a pipeline of a
method for performing a biological workflow for display to a user,
the pipeline of a method comprising a plurality of methods, each
method generating at least one biomolecule that may be used in the
next method to produce another biomolecule, wherein subroutines of
each of the plurality of methods are listed in a sequential order;
and executing the pipeline of the method.
25. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor of claim 1, further
comprising providing an option to display to the user, one or more
biomolecules resulting from reexecution of the subroutines of the
workflow.
26. The non-transitory computer-readable storage medium encoded
with instructions, executable by a processor of claim 1, further
comprising providing the ability to input data from a biological
instrument wherein the data is used during execution of a
subroutine.
27. A computer-implemented method comprising: presenting to a user
a plurality of subroutines that comprise the biological workflow
listed in a sequential order, wherein at least two subroutines of
the plurality of subroutines comprise two steps; providing the user
ability to navigate to any subroutine of the plurality of
sequential subroutines, to select a subroutine and view, set, or
change one or more parameters associated with a step of the
selected subroutine; providing an option to display to the user,
one or more biomolecules resulting from execution of one or more of
the plurality of subroutines; and providing an option to the user
to navigate to a prior subroutine and change a parameter of a step
of the prior subroutine, if the user is not satisfied with the one
or more displayed biomolecules.
28. A system comprising: a processor; and a memory for storing
instructions executable by the processor, the instructions
comprising instructions for: presenting to a user a plurality of
subroutines that comprise the biological workflow listed in a
sequential order, wherein at least two subroutines of the plurality
of subroutines comprise two steps; providing the user ability to
navigate to any subroutine of the plurality of sequential
subroutines, to select a subroutine and view, set, or change one or
more parameters associated with a step of the selected subroutine;
providing an option to display to the user, one or more
biomolecules resulting from execution of one or more of the
plurality of subroutines; and providing an option to the user to
navigate to a prior subroutine and change a parameter of a step of
the prior subroutine, if the user is not satisfied with the one or
more displayed biomolecules.
Description
CROSS REFERENCE TO OTHER APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
13/724,765 filed Dec. 21, 2012, which claims priority to U.S.
Provisional Patent Application Ser. No. 61/578,820, filed Dec. 21,
2011, the entire contents of which are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 4, 2013, is named LT00642_SL.txt and is 5,806 bytes in
size.
FIELD OF THE DISCLOSURE
[0003] The present disclosure is directed to bioinformatics,
especially to computer systems and computer software relating to
methods for in silico designing of and performing biological
workflows.
BACKGROUND
[0004] Biotechnology research that is important for improving
agricultural products, discovering new treatments for diseases, and
for identifying and developing new diagnostic methods, relies on
complex technologies, methods and experimental design. This
research would be greatly facilitated by computer assisted
experimental design programs.
SUMMARY OF THE DISCLOSURE
[0005] The present disclosure relates, in some embodiments, to a
non-transitory computer-readable storage medium encoded with
instructions, executable by a processor, the instructions
comprising: instructions for providing a method for performing a
biological workflow in silico comprising presenting to a user a
plurality of subroutines that comprise the biological workflow
listed in a sequential order, wherein at least two subroutines of
the plurality of subroutines comprise two steps; providing the user
ability to navigate to any subroutine of the plurality of
sequential subroutines, to select a subroutine and view, set, or
change one or more parameters associated with a step of the
selected subroutine; providing an option to display to the user,
one or more biomolecules resulting from execution of one or more of
the plurality of subroutines; and providing an option to the user
to navigate to a prior subroutine and change a parameter of a step
of the prior subroutine, if the user is not satisfied with the one
or more displayed biomolecules.
[0006] Non limiting examples of a biological workflow include
cloning methods, recombination methods, ligation methods, vector
designing methods, methods for synthesis of a nucleic acid, primer
design methods, methods for synthesis of a polypeptide, methods for
analysis of a cloned molecule, methods of protein analysis, methods
for making a modified host.
[0007] In some embodiments, instructions on a computer-readable
storage medium, according to the present disclosure, can further
comprise providing an indication to a user of a current subroutine
of the method for performing a biological workflow for the user to
review. Indications can also be provided to a user of a current
step and/or parameter being selected. In some embodiments, an
indication may comprise a highlighted tab in a GUI pane and/or a
flashing tab.
[0008] In some embodiments, a parameter being viewed, set or
changed is a parameter that results from ambiguous user data. In
some embodiments, one or all parameters that result from ambiguous
user data may be highlighted. Non-limiting examples of ambiguous
data includes "fuzzy ends" that occur when a DNA sequence cannot be
clearly read.
[0009] In some embodiments, instructions on a computer-readable
storage medium can further comprise receiving at least one
selection desired by a user, from the user, for one or more
parameters associated with at least one step of the plurality of
subroutines of the method for performing a biological workflow; and
providing to the user an option for storing in a memory a user
defined workflow comprising settings selected by the user for
parameters used in the biological workflow.
[0010] In some embodiments, instructions on the computer-readable
storage medium can further comprise providing to a user or a
plurality of users (community of users) the ability to rate or
comment on the user defined workflow and storing the rating
associated with the user defined workflow in the memory. This can
comprise receiving and storing from user(s) at least one comment
about the entire user defined workflow, and/or about one or more
subroutine(s) in a workflow, and/or about one or more step(s) in a
workflow and/or about one or more parameters in a workflow. In some
embodiments, instructions on the computer-readable storage medium
can further comprise, providing to a user, preset settings for
parameters based on user defined workflows with the highest ratings
or the most positive comments. In other words a computer readable
medium of the present disclosure can learn and suggest to a user
the best possible options for a parameter based in highest ratings
or positive comments to the parameter in that workflow from other
users.
[0011] In some embodiments, instructions on the computer-readable
storage medium can further comprise providing to a user the ability
to save a log of all selected parameters for each step of the user
defined method. In some examples this provides an electronic log
book or a lab notebook type of recording of all parameters, steps
that were user-defined or user selected for a workflow.
[0012] In some embodiments, one or more external files (that may be
related to the biological workflow) can be uploaded and stored by a
user and the computer-readable medium can have instructions for
providing additional users the ability to view the external files
uploaded.
[0013] Parameters viewed, selected, set or changed by a user can
comprise default parameters, which are pre-determined parameters
stored in the computer-readable storage medium, and/or user input
parameter, which are either modified default parameters, parameters
input by user, and/or a parameter imported by the user into the
computer system. In some embodiments one or more parameters viewed,
selected, set or changed by a user comprise a combination of one or
more default parameters and one or more user defined
parameters.
[0014] Providing a user ability to navigate to any subroutine of
the plurality of subroutines can be by a graphical user interface
(GUI) which can comprise displaying on a first display screen pane
all the subroutines of a sequential subroutine comprising the
biological workflow and, following selection by the user of any one
subroutine, displaying on a second display screen, one or more
steps associated with the selected subroutine. Providing the user
the ability to navigate to any step of a subroutine can also be
accomplished by a graphical user interface (GUI) which comprises
displaying on a first display screen pane a subroutine and
displaying on a second display screen, one or more steps associated
with the selected subroutine.
[0015] Selections received from a user of one or more parameters
for each step of the plurality of subroutines of the biological
workflow can be saved as a selected plurality of steps, which
comprise a user-defined method which can be saved and named by a
user.
[0016] In some embodiments, instructions on the computer-readable
storage medium can comprise: a) displaying on a display screen pane
a prescribed plurality of subroutines in a sequential order of the
biological workflow, wherein the prescribed plurality of
subroutines are comprised in a computer readable format; b)
navigation by a user using a graphical user interface (GUI) on the
display screen of each subroutine of the prescribed plurality of
subroutines of the biological workflow; c) selection by the user of
one of the subroutines; d) navigation by a user using a GUI on the
display screen of each step of a selected subroutine; e) selection
by the user of one or more parameters in each step of a selected
subroutine to obtain a modified plurality of steps; f) storing the
modified plurality of steps by the user, wherein the stored
modified plurality of steps comprise the user defined subroutine g)
repeating steps b)-f) till all the plurality of sub routines are
stored as user defined subroutines; and h) saving and executing the
plurality of user defined subroutines to perform a user defined
biological workflow.
[0017] In some embodiments, navigation by a user of any subroutine
or step of the workflow can be in a sequential order of the steps
or the subroutine. In some embodiments, navigation by a user of any
subroutine or step of the workflow can be in a non-sequential order
of the steps or the subroutine.
[0018] In some embodiments, a non-transitory computer-readable
storage medium encoded with instructions, executable by a processor
as described herein can further comprise: executing a user defined
workflow comprising executing in silico all the steps of the user
defined workflow in sequential order; viewing a first biomolecule
obtained by executing the user defined workflow in silico;
generation of at least a second user defined workflow, comprising
changing the selection of at least one parameter to have a
different value relative to the same parameter that was selected in
claim 1; executing in silico the at least second user defined
workflow to obtain a second biomolecule; viewing the second
biotechnology product in silico; and comparing the first
biomolecule with the second biomolecule, thereby allowing a user to
determine if the first user defined workflow or the second user
defined method produces a preferred biomolecule. This can be
repeated for several user-defined workflows, till the user finds an
optimum biomolecule and/or an optimum workflow to create a desired
biomolecule. In some embodiments, users may select a workflow based
on a biomolecule that is preferred.
[0019] In some embodiments a non-transitory computer-readable
storage medium encoded with instructions, executable by a
processor, comprises instructions for: providing a pipeline of a
method for performing a biological workflow for display to a user,
the pipeline of a method comprising a plurality of methods, each
method generating at least one biomolecule that may be used in the
next method to produce another biomolecule, wherein subroutines of
each of the plurality of methods are listed in a sequential order;
and executing the pipeline of the method. Additionally, a
non-transitory computer readable medium can further comprise
providing an option to display to a user, one or more biomolecules
resulting from reexecution of the subroutines of the workflow.
[0020] In some embodiments, a non-transitory computer readable
medium can further comprise providing the ability to input data
from a biological instrument wherein the data is used during
execution of a subroutine.
[0021] The present disclosure, in some embodiments also relates to
a computer-implemented method comprising: presenting to a user a
plurality of subroutines that comprise the biological workflow
listed in a sequential order, wherein at least two subroutines of
the plurality of subroutines comprise two steps; providing the user
ability to navigate to any subroutine of the plurality of
sequential subroutines, to select a subroutine and view, set, or
change one or more parameters associated with a step of the
selected subroutine; providing an option to display to the user,
one or more biomolecules resulting from execution of one or more of
the plurality of subroutines; and providing an option to the user
to navigate to a prior subroutine and change a parameter of a step
of the prior subroutine, if the user is not satisfied with the one
or more displayed biomolecules.
[0022] In some embodiments, the disclosure describes a system
comprising: a processor; and a memory for storing instructions
executable by the processor, the instructions comprising
instructions for: presenting to a user a plurality of subroutines
that comprise the biological workflow listed in a sequential order,
wherein at least two subroutines of the plurality of subroutines
comprise two steps; providing the user ability to navigate to any
subroutine of the plurality of sequential subroutines, to select a
subroutine and view, set, or change one or more parameters
associated with a step of the selected subroutine; providing an
option to display to the user, one or more biomolecules resulting
from execution of one or more of the plurality of subroutines; and
providing an option to the user to navigate to a prior subroutine
and change a parameter of a step of the prior subroutine, if the
user is not satisfied with the one or more displayed
biomolecules.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 illustrates a general computer system architecture
schematic according to an embodiment of the disclosure.
[0024] FIG. 2 is a block diagram that illustrates a computer system
700 that may be employed to carry out processing functionality,
according to some exemplary embodiments of the disclosure.
[0025] FIG. 3 depicts a flow chart of an example method 800
according to an embodiment of the present disclosure.
[0026] FIG. 4 depicts an example workflow method according to an
example embodiment of the present disclosure.
[0027] FIG. 5 depicts a flow chart of an example workflow method
according to an embodiment of the present disclosure.
[0028] FIG. 6 depicts an example workflow method according to an
example embodiment of the present disclosure.
[0029] FIGS. 7A-7O depicts a graphical user interface (GUI) in
accordance to one embodiment of a workflow method of the disclosure
wherein: FIG. 7A depicts a Gateway.RTM. Project GUI (HG 7C
discloses SEQ ID NOS: 1-2, respectively, in order of appearance);
FIG. 7B depicts a Task List GUI; FIG. 7C depicts a Current Task
GUI; FIG. 7D depicts a Gateway.RTM. Project GUI with options for
navigating to additional screens such as Saving, Editing and
Closing Projects; FIG. 7E depicts showing a Projects GUI; FIG. 7F
depicts a Fragments to Amplify GUI; FIG. 7G depicts a GUI when
Amplify button is selected (FIG. 7G discloses SEQ ID NO: 3); FIG.
7H depicts a Recombine Entry Clone by BP task pane GUI; FIG. 7I
depicts a pDONR.RTM. Vector which is an Gateway.RTM. Cloning
vector; FIG. 7J depicts a Preview Entry Clone task pane GUI; FIG.
7K depicts tools of a GUI for magnifying and zooming; FIG. 7L
depicts tools of the GUI to Display a Molecule as linear or
circular; FIG. 7M depicts a Create Expression Clones by LR task
pane GUI; FIG. 7N depicts a pDEST.RTM. Vector; and FIG. 7O depicts
GUI pane showing an example Preview Expression Clones task pane
showing an example expression clone created.
[0030] FIGS. 8A-8H depicts a graphical user interface (GUI) in
accordance to one embodiment of a workflow method of the disclosure
wherein: FIG. 8A depicts a GUI showing a TOPO.RTM. Project screen;
FIG. 8B depicts a GUI showing a Task List screen; FIG. 8C depicts a
GUI showing a Current Task screen; FIG. 8D depicts a GUI showing a
TOPO.RTM. Project screen with options for navigating to additional
screens such as Saving, Editing and Closing Projects; FIG. 8E
depicts a GUI showing a TOPO.RTM. Project screen showing a Projects
screen; FIG. 8F depicts a GUI showing a Fragments to Amplify in
TOPO.RTM. Reaction screen; FIG. 8G depicts a GUI showing a screen
when Amplify button is selected (FIG. 8G discloses SEQ ID NOS: 4-5,
respectively, in order of appearance); FIG. 8H depicts a GUI
showing a screen for Create TOPO.RTM. Clones task pane.
[0031] FIGS. 9A-9G depicts a graphical user interface (GUI) in
accordance to one embodiment of a workflow method of the disclosure
wherein: FIG. 9A depicts a GUI showing Launching ContigExpress.RTM.
screen; FIG. 9B depicts a GUI showing a ContigExpress.RTM. Project
screen with options for selecting ABI filed from a file system;
FIG. 9C depicts a GUI showing a View Fragments panel (FIG. 9C
discloses SEQ ID NO: 6); FIG. 9D depicts a GUI showing an example
screen for Trim Ends of all Fragments viewing (FIG. 9D discloses
SEQ ID NO: 7); FIG. 9E depicts a GUI screen showing a view of
specific details of ends trimming by selecting a fragment of
interest (FIG. 9E discloses SEQ ID NO: 6); FIG. 9F depicts a screen
of Contig assembly parameters; and FIG. 9 G depicts an example
screen viewing Resultant Contig (FIG. 9G discloses SEQ ID NOS: 8,
8, 13 AND 13-14, respectively, in order of appearance).
[0032] FIG. 10 shows an illustration of a typical internet network
configuration where a number of client machines, possibly in a
remote local office, are shown connected to a
gateway/hub/tunnel-server/etc.
[0033] Additional figures and figure explanations are provided
within the illustrative examples provided herein.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0034] In the description that follows, a number of terms used in
recombinant nucleic acid technology are utilized extensively. In
order to provide a clear and more consistent understanding of the
specification and claims, including the scope to be given such
terms, the following definitions are provided.
[0035] Genomic Products and Services: As used herein, the term
genomic products and services refers to products and services that
may be used to conduct research involving nucleic acids, including
RNA interference (RNAi).
[0036] Proteomic Products and Services: As used herein, the term
proteomic products and services refers to products and services
that may be used to conduct research involving polypeptides.
[0037] Clone Collection: As used herein, "clone collection" refers
to two or more nucleic acid molecules, each of which comprises one
or more nucleic acid sequences of interest.
[0038] Customer: As used herein, the term customer refers to any
individual, institution, corporation, university, or organization
seeking to obtain genomic and proteomic products and services.
[0039] Provider: As used herein, the term provider refers to any
individual, institution, corporation, university, or organization
seeking to provide genomic and proteomic products and services.
[0040] Subscriber: As used herein, the term subscriber refers to
any customer having an agreement with a provider to obtain public
and private genomic and proteomic products and services at
subscriber rates.
[0041] Non-subscriber: As used herein, the term non-subscriber
refers to any customer who does not have an agreement with a
provider to obtain public and private genomic and proteomic
products and services at subscriber rates.
[0042] Host: As used herein, the term "host" refers to any
prokaryotic or eukaryotic (e.g., mammalian, insect, yeast, plant,
avian, animal, etc.) cell and/or organism that is a recipient of a
replicable expression vector, cloning-vector or any nucleic acid
molecule. The nucleic acid molecule may contain, but is not limited
to, a sequence of interest, a transcriptional regulatory sequence
(such as a promoter, enhancer, repressor, and the like) and/or an
origin of replication. As used herein, the terms "host," "host
cell," "recombinant host" and "recombinant host cell" may be used
interchangeably. For examples of such hosts, see Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
[0043] Transcriptional Regulatory Sequence: As used herein, the
phrase "transcriptional regulatory sequence" refers to a functional
stretch of nucleotides contained on a nucleic acid molecule, in any
configuration or geometry, that act to regulate the transcription
of (1) one or more nucleic acid sequences that may comprise ORFs,
(e.g., two, three, four, five, seven, ten, etc.) into messenger RNA
or (2) one or more nucleic acid sequences into untranslated RNA.
Examples of transcriptional regulatory sequences include, but are
not limited to, promoters, enhancers, repressors, operators (e.g.,
the tet operator), and the like.
[0044] Promoter: As used herein, a promoter is an example of a
transcriptional regulatory sequence, and is specifically a nucleic
acid generally described as the 5'-region of a gene located
proximal to the start codon or nucleic acid that encodes
untranslated RNA. The transcription of an adjacent nucleic acid
segment is initiated at or near the promoter. A repressible
promoter's rate of transcription decreases in response to a
repressing agent. An inducible promoter's rate of transcription
increases in response to an inducing agent. A constitutive
promoter's rate of transcription is not specifically regulated,
though it can vary under the influence of general metabolic
conditions.
[0045] Insert: As used herein, the term "insert" refers to a
desired nucleic acid segment that is a part of a larger nucleic
acid molecule. In many instances, the insert will be introduced
into the larger nucleic acid molecule using techniques known to
those of skill in the art, e.g., recombinational cloning,
topoisomerase cloning or joining, ligation, etc.
[0046] Target Nucleic Acid Molecule: As used herein, the phrase
"target nucleic acid molecule" refers to a nucleic acid molecule
comprising at least one nucleic acid sequence of interest,
preferably a nucleic acid molecule that is to be acted upon using
the compounds and methods of the present disclosure. Such target
nucleic acid molecules may contain one or more (e.g., two, three,
four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty,
etc.) sequences of interest.
[0047] Recognition Sequence: As used herein, the phrase
"recognition sequence" or "recognition site" refers to a particular
sequence to which a protein, chemical compound, DNA, or RNA
molecule (e.g., restriction endonuclease, a topoisomerase, a
modification methylase, a recombinase, etc.) recognizes and binds.
In the present disclosure, a recognition sequence may refer to a
recombination site. For example, the recognition sequence for Cre
recombinase is loxP which is a 34 base pair sequence comprising two
13 base pair inverted repeats (serving as the recombinase binding
sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer,
B., Current Opinion in Biotechnology 5:521-527 (1994)). Other
examples of recognition sequences are the attB, attP, attL, and
attR sequences, which are recognized by the recombinase enzyme X
Integrase. attB is an approximately 25 base pair sequence
containing two 9 base pair core-type Int binding sites and a 7 base
pair overlap region. attP is an approximately 240 base pair
sequence containing core-type Int binding sites and arm-type Int
binding sites as well as sites for auxiliary proteins integration
host factor (IHF), FIS and excisionase (Xis) (see Landy, Current
Opinion in Biotechnology 3:699-707 (1993)). Such sites may also be
engineered according to the present disclosure to enhance
production of products, such as biomolecules, in the methods of the
disclosure. For example, when such engineered sites lack the P1 or
HI domains to make the recombination reactions irreversible (e.g.,
attR or attP), such sites may be designated attR' or attP' to show
that the domains of these sites have been modified in some way.
[0048] Recombination Proteins: As used herein, the phrase
"recombination proteins" includes excisive or integrative proteins,
enzymes, co-factors or associated proteins that are involved in
recombination reactions involving one or more recombination sites
(e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty,
thirty, fifty, etc.), which may be wild-type proteins (see Landy,
Current Opinion in Biotechnology 3:699-707 (1993)), or mutants,
derivatives (e.g., fusion proteins containing the recombination
protein sequences or fragments thereof), fragments, and variants
thereof. Examples of recombination proteins include Cre, Int, IHF,
Xis, Flp, Fis, Hin, Gin, .PHI.C31, Cin, Tn3 resolvase, TndX, XerC,
XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.
[0049] Recombinases: As used herein, the term "recombinases" is
used to refer to the protein that catalyzes strand cleavage and
re-ligation in a recombination reaction. Site-specific recombinases
are proteins that are present in many organisms (e.g., viruses and
bacteria) and have been characterized as having both endonuclease
and ligase properties. These recombinases (along with associated
proteins in some cases) recognize specific sequences of bases in a
nucleic acid molecule and exchange the nucleic acid segments
flanking those sequences. The recombinases and associated proteins
are collectively referred to as "recombination proteins" (see,
e.g., Landy, A., Current Opinion in Biotechnology 3:699-707
(1993)).
[0050] Numerous recombination systems from various organisms have
been described. See, e.g., Hoess, et al., Nucleic Acids Research
14(6):2287 (1986); Abremski, et al., J. Biol. Chem. 261(1):391
(1986); Campbell, J. Bacteriol. 174(23):7495 (1992); Qian, et al.,
J. Biol. Chem. 267(11):7794 (1992); Araki, et al., J. Mol. Biol.
225(1):25 (1992); Maeser and Kahnmann, Mol. Gen. Genet. 230:170-176
(1991); Esposito, et al., Nucl. Acids Res. 25(18):3605 (1997). Many
of these belong to the integrase family of recombinases (Argos, et
al., EMBO J. 5:433-440 (1986); Voziyanov, et al., Nucl. Acids Res.
27:930 (1999)). Perhaps the best studied of these are the
Integrase/att system from bacteriophage.lamda. (Landy, A. Current
Opinions in Genetics and Devel. 3:699-707 (1993)), the Cre/loxP
system from bacteriophage P1 (Hoess and Abremski (1990) In Nucleic
Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,
Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT
system from the Saccharomyces cerevisiae 2 .mu.circle plasmid
(Broach, et al., Cell 29:227-234 (1982)).
[0051] Recombination Site: A used herein, the phrase "recombination
site" refers to a recognition sequence on a nucleic acid molecule
that participates in an integration/recombination reaction by
recombination proteins. Recombination sites are discrete sections
or segments of nucleic acid on the participating nucleic acid
molecules that are recognized and bound by a site-specific
recombination protein during the initial stages of integration or
recombination. For example, the recombination site for Cre
recombinase is loxP, which is a 34 base pair sequence comprised of
two 13 base pair inverted repeats (serving as the recombinase
binding sites) flanking an 8 base pair core sequence (see FIG. 1 of
Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Other examples
of recombination sites include the attB, attP, attL, and attR
sequences described in U.S. provisional patent applications
60/136,744, filed May 28, 1999, and 60/188,000, filed Mar. 9, 2000,
and in U.S. patent application Ser. Nos. 09/517,466 and
09/732,91--all of which are specifically incorporated herein by
reference--and mutants, fragments, variants and derivatives
thereof, which are recognized by the recombination protein .lamda.
Int and by the auxiliary proteins integration host factor (IHF),
FIS and excisionase (Xis) (see Landy, Curr. Opin. Biotech.
3:699-707 (1993)).
[0052] Mutating specific residues in the core region of the att
site can generate a large number of different att sites. As with
the att I and att2 sites utilized in GATEWAY.TM., each additional
mutation potentially creates a novel att site with unique
specificity that will recombine only with its cognate partner att
site bearing the same mutation and will not cross-react with any
other mutant or wild-type att site. Novel mutated att sites (e.g.,
attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described in
previous patent application Ser. No. 09/517,466, filed Mar. 2,
2000, which is specifically incorporated herein by reference. Other
recombination sites having unique specificity (i.e., a first site
will recombine with its corresponding site and will not recombine
or not substantially recombine with a second site having a
different specificity) may be used to practice the present
disclosure. Examples of suitable recombination sites include, but
are not limited to, loxP sites; loxP site mutants, variants or
derivatives such as loxP511 (see U.S. Pat. No. 5,851,808); frt
sites; frt site mutants, variants or derivatives; dif sites; dif
site mutants, variants or derivatives; psi sites; psi site mutants,
variants or derivatives; cer sites; and cer site mutants, variants
or derivatives.
[0053] Recombination sites may be added to molecules by any number
of known methods. For example, recombination sites can be added to
nucleic acid molecules by blunt end ligation, PCR performed with
fully or partially random primers, or inserting the nucleic acid
molecules into a vector using a restriction site flanked by
recombination sites.
[0054] Recombinational Cloning: As used herein, the phrase
"recombinational cloning" refers to a method whereby segments of
nucleic acid molecules or populations of such molecules are
exchanged, inserted, replaced, substituted or modified, in vitro or
in vivo. Preferably, such cloning method is an in vitro method.
[0055] Suitable recombinational cloning systems that utilize
recombination at defined recombination sites have been previously
described in U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861,
6,270,969, and 6,277,608, and in pending U.S. application Ser. No.
09/517,466, and in published United States application no.
20020007051, (each of which is fully incorporated herein by
reference), all assigned to the Invitrogen Corporation, Carlsbad,
Calif. In brief, the GATEWAY.TM. Cloning System described in these
patents utilizes vectors that contain at least one recombination
site to clone desired nucleic acid molecules in vivo or in vitro.
In some embodiments, the system utilizes vectors that contain at
least two different site-specific recombination sites that may be
based on the bacteriophage lambda system (e.g., att1 and att2) that
are mutated from the wild-type (att0) sites. Each mutated site has
a unique specificity for its cognate partner att site (i.e., its
binding partner recombination site) of the same type (for example
attB1 with attP1, or attL1 with attR1) and will not cross-react
with recombination sites of the other mutant type or with the
wild-type att0 site. Different site specificities allow directional
cloning or linkage of desired molecules thus providing desired
orientation of the cloned molecules. Nucleic acid fragments flanked
by recombination sites are cloned and subcloned using the
GATEWAY.TM. system by replacing a selectable marker (for example,
ccdB) flanked by att sites on the recipient plasmid molecule,
sometimes termed the Destination Vector. Desired clones are then
selected by transformation of a ccdB sensitive host strain and
positive selection for a marker on the recipient molecule. Similar
strategies for negative selection (e.g., use of toxic genes) can be
used in other organisms such as thymidine kinase (TK) in mammals
and insects.
[0056] Topoisomerase recognition site: As used herein, the term
"topoisomerase recognition site" means a defined nucleotide
sequence that is recognized and bound by a site specific
topoisomerase. For example, the nucleotide sequence 5'-(C/T)CCTT-3'
is a topoisomerase recognition site that is bound specifically by
most poxvirus topoisomerases, including vaccinia virus DNA
topoisomerase I, which then can cleave the strand after the 3'-most
thymidine of the recognition site to produce a nucleotide sequence
comprising 5'-(C/T)CCTT-PO.sub.4-TOPO, i.e., a complex of the
topoisomerase covalently bound to the 3' phosphate through a
tyrosine residue in the topoisomerase (see, Shuman, J. Biol. Chem.
266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res.
22:5360-5365, 1994; each of which is incorporated herein by
reference; see, also, U.S. Pat. No. 5,766,891; PCT/US95/16099; and
PCT/US98/12372). In comparison, the nucleotide sequence
5'-GCAACTT-3' is the topoisomerase recognition site for type IA E.
coli topoisomerase III.
[0057] Repression Cassette: As used herein, the phrase "repression
cassette" refers to a nucleic acid segment that contains a
repressor or a selectable marker present in the subcloning
vector.
[0058] Selectable Marker: As used herein, the phrase "selectable
marker" refers to a nucleic acid segment that allows one to select
for or against a molecule (e.g., a replicon) or a cell that
contains it, often under particular conditions. These markers can
encode an activity, such as, but not limited to, production of RNA,
peptide, or protein, or can provide a binding site for RNA,
peptides, proteins, inorganic and organic compounds or compositions
and the like. Examples of selectable markers include but are not
limited to: (1) nucleic acid segments that encode products that
provide resistance against otherwise toxic compounds (e.g.,
antibiotics); (2) nucleic acid segments that encode products that
are otherwise lacking in the recipient cell (e.g., tRNA genes,
auxotrophic markers); (3) nucleic acid segments that encode
products that suppress the activity of a gene product; (4) nucleic
acid segments that encode products that can be readily identified
(e.g., phenotypic markers such as (beta-galactosidase, green
fluorescent protein (GFP), yellow flourescent protein (YFP), red
fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell
surface proteins); (5) nucleic acid segments that bind products
that are otherwise detrimental to cell survival and/or function;
(6) nucleic acid segments that otherwise inhibit the activity of
any of the nucleic acid segments described in Nos. 1-5 above (e.g.,
antisense oligonucleotides); (7) nucleic acid segments that bind
products that modify a substrate (e.g., restriction endonucleases);
(8) nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g., specific protein binding sites); (9)
nucleic acid segments that encode a specific nucleotide sequence
that can be otherwise non-functional (e.g., for PCR amplification
of subpopulations of molecules); (10) nucleic acid segments that,
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; and/or (11) nucleic acid
segments that encode products that either are toxic (e.g.,
Diphtheria toxin) or convert a relatively non-toxic compound to a
toxic compound (e.g., Herpes simplex thymidine kinase, cytosine
deaminase) in recipient cells; (12) nucleic acid segments that
inhibit replication, partition or heritability of nucleic acid
molecules that contain them; and/or (13) nucleic acid segments that
encode conditional replication functions, e.g., replication in
certain hosts or host cell strains or under certain environmental
conditions (e.g., temperature, nutritional conditions, etc.).
[0059] Site-Specific Recombinase: As used herein, the phrase
"site-specific recombinase" refers to a type of recombinase that
typically has at least the following four activities (or
combinations thereof): (1) recognition of specific nucleic acid
sequences; (2) cleavage of said sequence or sequences; (3)
topoisomerase activity involved in strand exchange; and (4) ligase
activity to reseal the cleaved strands of nucleic acid (see Sauer,
B., Current Opinions in Biotechnology 5:521-527 (1994)).
Conservative site-specific recombination is distinguished from
homologous recombination and transposition by a high degree of
sequence specificity for both partners. The strand exchange
mechanism involves the cleavage and rejoining of specific nucleic
acid sequences in the absence of DNA synthesis (Landy, A. (1989)
Ann. Rev. Biochem. 58:913-949).
[0060] Suppressor tRNAs: As used herein, the phrase "suppressor
tRNA" refers to a molecule that mediates the incorporation of an
amino acid in a polypeptide in a position corresponding to a stop
codon in the mRNA being translated.
[0061] Homologous Recombination: As used herein, the phrase
"homologous recombination" refers to the process in which nucleic
acid molecules with similar nucleotide sequences associate and
exchange nucleotide strands. A nucleotide sequence of a first
nucleic acid molecule that is effective for engaging in homologous
recombination at a predefined position of a second nucleic acid
molecule will therefore have a nucleotide sequence that facilitates
the exchange of nucleotide strands between the first nucleic acid
molecule and a defined position of the second nucleic acid
molecule. Thus, the first nucleic acid will generally have a
nucleotide sequence that is sufficiently complementary to a portion
of the second nucleic acid molecule to promote nucleotide base
pairing.
[0062] Homologous recombination: requires homologous sequences in
the two recombining partner nucleic acids but does not require any
specific sequences. As indicated above, site-specific recombination
that occurs, for example, at recombination sites such as att sites,
is not considered to be "homologous recombination," as the phrase
is used herein.
[0063] Vector: As used herein, the term "vector" refers to a
nucleic acid molecule (preferably DNA) that provides a useful
biological or biochemical property to an insert. Examples include
plasmids, phages, viruses, autonomously replicating sequences
(ARS), centromeres, and other sequences that are able to replicate
or be replicated in vitro or in a host cell, or to convey a desired
nucleic acid segment to a desired location within a host cell. A
vector can have one or more restriction endonuclease recognition
sites (e.g., two, three, four, five, seven, ten, etc.) at which the
sequences can be cut in a determinable fashion without loss of an
essential biological function of the vector, and into which a
nucleic acid fragment can be spliced in order to bring about its
replication and cloning. Vectors can further provide primer sites
(e.g., for PCR), transcriptional and/or translational initiation
and/or regulation sites, recombinational signals, replicons,
selectable markers, etc. Clearly, methods of inserting a desired
nucleic acid fragment that do not require the use of recombination,
transpositions or restriction enzymes (such as, but not limited to,
uracil N-glycosylase (UDG) cloning of PCR fragments (U.S. Pat. Nos.
5,334,575 and 5,888,795, both of which are entirely incorporated
herein by reference), T:A cloning, and the like) can also be
applied to clone a fragment into a cloning vector to be used
according to the present disclosure. The cloning vector can further
contain one or more selectable markers (e.g., two, three, four,
five, seven, ten, etc.) suitable for use in the identification of
cells transformed with the cloning vector.
[0064] Subcloning Vector: As used herein, the phrase "subcloning
vector" refers to a cloning vector comprising a circular or linear
nucleic acid molecule that includes, preferably, an appropriate
replicon. In the present disclosure, the subcloning vector can also
contain functional and/or regulatory elements that are desired to
be incorporated into the final product to act upon or with the
cloned nucleic acid insert. The subcloning vector can also contain
a selectable marker (preferably DNA).
[0065] Primer: As used herein, the term "primer" refers to a single
stranded or double stranded oligonucleotide that is extended by
covalent bonding of nucleotide monomers during amplification or
polymerization of a nucleic acid molecule (e.g., a DNA molecule).
In one aspect, the primer may be a sequencing primer (for example,
a universal sequencing primer). In another aspect, the primer may
comprise a recombination site or portion thereof.
[0066] Adapter: As used herein, the term "adapter" refers to an
oligonucleotide or nucleic acid fragment or segment (preferably
DNA) that comprises one or more recombination sites (or portions of
such recombination sites) that can be added to a circular or linear
nucleic acid molecule as well as to other nucleic acid molecules
described herein. When using portions of recombination sites, the
missing portion may be provided by the nucleic acid molecule. Such
adapters may be added at any location within a circular or linear
molecule, although the adapters are preferably added at or near one
or both termini of a linear molecule. Preferably, adapters are
positioned to be located on both sides (flanking) a particular
nucleic acid molecule of interest. In accordance with the
disclosure, adapters may be added to nucleic acid molecules of
interest by standard recombinant techniques (e.g., restriction
digest and ligation). For example, adapters may be added to a
circular molecule by first digesting the molecule with an
appropriate restriction enzyme, adding the adapter at the cleavage
site and reforming the circular molecule that contains the
adapter(s) at the site of cleavage. In other aspects, adapters may
be added by homologous recombination, by integration of RNA
molecules, and the like. Alternatively, adapters may be ligated
directly to one or more and preferably both termini of a linear
molecule thereby resulting in linear molecule(s) having adapters at
one or both termini. In one aspect of the disclosure, adapters may
be added to a population of linear molecules, (e.g., a cDNA library
or genomic DNA that has been cleaved or digested) to form a
population of linear molecules containing adapters at one and
preferably both termini of all or substantial portion of said
population.
[0067] Adapter-Primer: As used herein, the phrase "adapter-primer"
refers to a primer molecule that comprises one or more
recombination sites (or portions of such recombination sites) that
can be added to a circular or to a linear nucleic acid molecule
described herein. When using portions of recombination sites, the
missing portion may be provided by a nucleic acid molecule (e.g.,
an adapter) of the disclosure. Such adapter-primers may be added at
any location within a circular or linear molecule, although the
adapter-primers are preferably added at or near one or both termini
of a linear molecule. Such adapter-primers may be used to add one
or more recombination sites or portions thereof to circular or
linear nucleic acid molecules in a variety of contexts and by a
variety of techniques, including but not limited to amplification
(e.g., PCR), ligation (e.g., enzymatic or chemical/synthetic
ligation), recombination (e.g., homologous or non-homologous
(illegitimate) recombination) and the like.
[0068] Template: As used herein, the term "template" refers to a
double stranded or single stranded nucleic acid molecule, all or a
portion of which is to be amplified, synthesized, reverse
transcribed, or sequenced. In the case of a double-stranded DNA
molecule, denaturation of its strands to form a first and a second
strand is preferably performed before these molecules may be
amplified, synthesized or sequenced, or the double stranded
molecule may be used directly as a template. For single stranded
templates, a primer complementary to at least a portion of the
template hybridizes under appropriate conditions and one or more
polypeptides having polymerase activity (e.g., two, three, four,
five, or seven DNA polymerases and/or reverse transcriptases) may
then synthesize a molecule complementary to all or a portion of the
template. Alternatively, for double stranded templates, one or more
transcriptional regulatory sequences (e.g., two, three, four, five,
seven or more promoters) may be used in combination with one or
more polymerases to make nucleic acid molecules complementary to
all or a portion of the template. The newly synthesized molecule,
according to the disclosure, may be of equal or shorter length
compared to the original template. Mismatch incorporation or strand
slippage during the synthesis or extension of the newly synthesized
molecule may result in one or a number of mismatched base pairs.
Thus, the synthesized molecule need not be exactly complementary to
the template. Additionally, a population of nucleic acid templates
may be used during synthesis or amplification to produce a
population of nucleic acid molecules typically representative of
the original template population.
[0069] Incorporating: As used herein, the term "incorporating"
means becoming a part of a nucleic acid (e.g., DNA) molecule or
primer.
[0070] Library: As used herein, the term "library" refers to a
collection of nucleic acid molecules (circular or linear). In one
embodiment, a library may comprise a plurality of nucleic acid
molecules (e.g., two, three, four, five, seven, ten, twelve,
fifteen, twenty, thirty, fifty, one hundred, two hundred, five
hundred one thousand, five thousand, or more), that may or may not
be from a common source organism, organ, tissue, or cell. In
another embodiment, a library is representative of all or a:
portion or a significant portion of the nucleic acid content of an
organism (a "genomic" library), or a set of nucleic acid molecules
representative of all or a portion or a significant portion of the
expressed nucleic acid molecules (a cDNA library or segments
derived therefrom) in a cell, tissue, organ or organism. A library
may also comprise nucleic acid molecules having random sequences
made by de novo synthesis, mutagenesis of one or more nucleic acid
molecules, and the like. Such libraries may or may not be contained
in one or more vectors (e.g., two, three, four, five, seven, ten,
twelve, fifteen, twenty, thirty, fifty, etc.). In some embodiments,
a library may be "normalized" library (i.e., a library of cloned
nucleic acid molecules from which each member nucleic acid molecule
can be isolated with approximately equivalent probability).
[0071] Normalized: As used herein, the term "normalized" or
"normalized library" means a nucleic acid library that has been
manipulated, preferably using the methods of the disclosure, to
reduce the relative variation in abundance among member nucleic
acid molecules in the library to a range of no greater than about
25-fold, no greater than about 20-fold, no greater than about
15-fold, no greater than about 10-fold, no greater than about
7-fold, no greater than about 6-fold, no greater than about 5-fold,
no greater than about 4-fold, no greater than about 3-fold or no
greater than about 2-fold.
[0072] Amplification: As used herein, the term "amplification"
refers to any in vitro method for increasing the number of copies
of a nucleic acid molecule with the use of one or more polypeptides
having polymerase activity (e.g., one, two, three, four or more
nucleic acid polymerases or reverse transcriptases). Nucleic acid
amplification results in the incorporation of nucleotides into a
DNA and/or RNA molecule or primer thereby forming a new nucleic
acid molecule complementary to a template. The formed nucleic acid
molecule and its template can be used as templates to synthesize
additional nucleic acid molecules. As used herein, one
amplification reaction may consist of many rounds of nucleic acid
replication. DNA amplification reactions include, for example,
polymerase chain reaction (PCR). One PCR reaction may consist of 5
to 100 cycles of denaturation and synthesis of a DNA molecule.
[0073] Nucleotide: As used herein, the term "nucleotide" refers to
a base-sugar-phosphate combination. Nucleotides are monomeric units
of a nucleic acid molecule (DNA and RNA). The term nucleotide
includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and
deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP,
dGTP, dTTP, or derivatives thereof. Such derivatives include, for
example, [.alpha.-S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term
nucleotide as used herein also refers to dideoxyribonucleoside
triphosphates (ddNTPs) and their derivatives. Illustrated examples
of dideoxyribonucleoside triphosphates include, but are not limited
to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present
disclosure, a "nucleotide" may be unlabeled or detectably labeled
by well known techniques. Detectable labels include, for example,
radioactive isotopes, fluorescent labels, chemiluminescent labels,
bioluminescent labels and enzyme labels.
[0074] Nucleic Acid Molecule: As used herein, the phrase "nucleic
acid molecule" refers to a sequence of contiguous nucleotides
(riboNTPs, dNTPs, ddNTPs, or combinations thereof) of any length. A
nucleic acid molecule may encode a full-length polypeptide or a
fragment of any length thereof, or may be non-coding. As used
herein, the terms "nucleic acid molecule" and "polynucleotide" may
be used interchangeably and include both RNA and DNA.
[0075] Oligonucleotide: As used herein, the term "oligonucleotide"
refers to a synthetic or natural molecule comprising a covalently
linked sequence of nucleotides that are joined by a phosphodiester
bond between the 3' position of the pentose of one nucleotide and
the 5' position of the pentose of the adjacent nucleotide. \
[0076] Open Reading Frame (ORF): As used herein, an open reading
frame or ORF refers to a sequence of nucleotides that codes for a
contiguous sequence of amino acids. ORFs of the disclosure may be
constructed to code for the amino acids of a polypeptide of
interest from the N-terminus of the polypeptide (typically a
methionine encoded by a sequence that is transcribed as AUG) to the
C-terminus of the polypeptide. ORFs of the disclosure include
sequences that encode a contiguous sequence of amino acids with no
intervening sequences (e.g., an ORF from a cDNA) as well as ORFs
that comprise one or more intervening sequences (e.g., introns)
that may be processed from an mRNA containing them (e.g., by
splicing) when an mRNA containing the ORF is transcribed in a
suitable host cell. ORFs of the disclosure also comprise splice
variants of ORFs containing intervening sequences.
[0077] ORFs may optionally be provided with one or more sequences
that function as stop codons (e.g., contain nucleotides that are
transcribed as UAG, an amber stop codon, UGA, an opal stop codon,
and/or UAA, an ochre stop codon). When present, a stop codon may be
provided after the codon encoding the C-terminus of a polypeptide
of interest (e.g., after the last amino acid of the polypeptide)
and/or may be located within the coding sequence of the polypeptide
of interest. When located after the C-terminus of the polypeptide
of interest, a stop codon may be immediately adjacent to the codon
encoding the last amino acid of the polypeptide or there may be one
or more codons (e.g., one, two, three, four, five, ten, twenty,
etc) between the codon encoding the last amino acid of the
polypeptide of interest and the stop codon. A nucleic acid molecule
containing an ORF may be provided with a stop codon upstream of the
initiation codon (e.g., an AUG codon) of the ORF. When located
upstream of the initiation codon of the polypeptide of interest, a
stop codon may be immediately adjacent to the initiation codon or
there may be one or more codons (e.g., one, two, three, four, five,
ten, twenty, etc) between the initiation codon and the stop
codon.
[0078] Polypeptide: As used herein, the term "polypeptide" refers
to a sequence of contiguous amino acids of any length. The terms
"peptide," "oligopeptide," or "protein" may be used interchangeably
herein with the term "polypeptide."
[0079] Hybridization: As used herein, the terms "hybridization" and
"hybridizing" refer to base pairing of two complementary
single-stranded nucleic acid molecules (RNA and/or DNA) to give a
double stranded molecule. As used herein, two nucleic acid
molecules may hybridize, although the base pairing is not
completely complementary. Accordingly, mismatched bases do not
prevent hybridization of two nucleic acid molecules provided that
appropriate conditions, well known in the art, are used. In some
aspects, hybridization is said to be under "stringent conditions."
By "stringent conditions," as the phrase is used herein, is meant
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0080] Feature: As used herein, the term "feature" refers to a
segment of a biomolecule that provides a specific function. For
example, a "feature" can be a region of a polypeptide or
polynucleotide that has a specific function. In an illustrative
example, a feature is a region of a vector that has a specific
function. For example, a feature on a vector includes, but is not
limited to, a restriction enzyme site, a recombination site, or a
tag-encoding sequence.
[0081] An exemplary list of vectors that can be used in the in
silico design methods, includes the following: BaculoDirect Linear
DIMA; BacuiloDirect Linear; DNA Cloning Fragment DNA; BaculoDirect
N-term Linear DNA_verA; BaculoDirect.TM. C-Term Baculovirus Linear
DNA; BaculoDirect.TM. N-Term Baculovirus Linear DNA; Champion.TM.
pET100/D-TOPO.RTM.; Champion.TM. pET 101/D-TOPO.RTM.; Champion.TM.
pET 102/D-TOPO.RTM.; Champion.TM. pET 104/D-TOPO.RTM.; Champion.TM.
pET104-DEST; Champion.TM. pET151/D-TOPO.COPYRGT; Champion.TM. pET
160/D-TOPO.RTM.; Champion.TM. pET 160-DEST; Champion.TM. pET
161-DEST; Champion.TM. pET200/D-TOPO.RTM.; pAc5.1N5-His A, B, and
C; pAd/BLOCK-iT-DEST; pAd/BLOCK-f!.''-DEST_verA_sz; pAd/CMVA/5
DEST; pAd/PL-DEST; pAO815; pBAD/glll A, B, and C; pBAD/His A, B,
and C; pBAD/myc-His A, B, and C; pBAD/Thio-TOPO.RTM.; pBAD
102/D-TOPO.RTM.; pBAD20/D-TOPO.RTM.; pBAD202/D-TOPO.RTM.; pBAD
DEST49; PBAD-TOPO; PBAD-TOPO.RTM.; pBC1; pBLOCK-ff3-DEST
pBLOCK-iT6-DEST pBlueBac4.5 pBlueBac4.5A/5-His TOPO.RTM.;
pBlueBacHis2 A, B, and C; pBR322; pBudCE4.1; pcDN3.1A/5-His-TOPO;
pcDNA3.1(-); pcDNA3.1(+); pcDNA3.1(+)/myc-HisA; pcDNA3.1(+)/myc-His
A, B, C; pcDNA3.1(+)/myc-His B; pcDNA3.1(+)/myc-HisC;
DCDNA3.1/CT-GFP-TOPO; pcDNA3.1/His A; pcDNA3.1/His B; pcDNA3.1/His
C; pcDNA3.1/Hygro(-); pcDNA3.1/Hygro(+); pcDNA3.1/NT-GFP-TOPO;
pcDNA3.1/nV5-DEST; pcDNA3.1A/5-His A; pcDNA3.1A/5-His B;
pcDNA3.1A/5-His C; pcDNA3.1/Zeo(-); pcDNA3.1/Zeo(+);
pcDNA3.1/Zeo(+); pcDNA3.1DA/5-His-TOPO; pcDNA3.2N5-DEST;
pcDNA3.2A/5-GW/D-TOPO; pcDNA3.2-DEST; pcDNA4/His A; pcDNA4/His B;
pcDNA4/His C; pcDNA4/HisMAX A, B & C; pcDNA4/HisMax-TOPO;
pcDNA4/HisMax-TOPO; pcDNA4/myc-His A, B, and C; pcDNA4/TO;
pcDNA4/TO; pcDNA4/TO/myc-His A; pcDNA4/TO/myc-His A, B, C;
pcDNA4/TO/myc-His B; pcDNA4/TO/myc-His C; pcDNA4N5-His A, B, and C;
pcDNA5/FRT; pcDNA5/FRT; pcDNA5/FRT/TO/CAT; pcDNA5/FRT/TO-TOPO;
pcDNA5/FRT/V5-His-TOPO; pcDNA5/TO;
pcDNA6.2/cGeneBLAzer-DEST_verA_sz; pcDNA6 2/cGeneBLAzer-GW/D-TOPO
pcDNA6; 2/cGeneBlazer-GW/D-TOPO_verA_sz pcDNA6.2/cLumio-DEST;
pcDNA6 2/cLumio-DE STverAsz pcDNA6.2/GFP-DEST_verA_sz;
pcDNA6.2/nGeneBLAzer-DEST pcDNA6 2/nGeneBLAzer-DEST_verA_sz pcDMA6
2/nGeneBlazer-GW/D-TOPO_verA_s2 pcDNA6.2/nLumio-DEST; pcDNA6
2/nLumio-DEST_verB_sz; pcDNA6.2A/5-DEST pcDNA6.2A/5-GW/D-TOPO
pcDNA6/BioEase-DEST verAsz; pcDNA6/H62His A, B, and C pcDNA6/His A,
B, and C; pcDNA6/TR; pcDNA6/V5-His A; pcDNA6/V5-His B;
pcDNA6/V5-His C; pcDNA6/V5-His C; pcDNA-DEST40; pcDNA-DEST47;
pcDNA-DEST53; pCEP4; pCEP4/CAT; pCMV/myc/cyto; pCMV/myc/ER;
pCMV/myc/mito; pCMV/myc/nuc; pCMVSPORT6 Notl-Sall Cut; pCoBlasi;
pCR Blunt; pCR XL TOPO; pCR.RTM. T7/CT TOPO.RTM.; pCR.RTM. T7/NT
TOPO.RTM.; pCR2.1-TOPO; pCR3.1; pCR3.1-Uni; pCR4BLUNT-TOPO;
pCR4-TOPO; pCR8/GW/TOPO TA; pCR8/GW-TOPO_verA_sz; pCR-Blunt
II-TOPO;-pCRII-TOPO; pDEST.TM. R4-R3; PDEST.TM. 10; PDEST.TM. 14;
PDEST.TM. 15; pDEST.TM. 17; pDEST.TM. 20; pDEST.TM.22; PDEST.TM.
24; pDEST.TM. 26; pDES.TM.27; pDEST.TM. 32; pDEST.TM.8; pDEST.TM.
38; pDEST.TM. 39; pDisplay; pDONR.TM. P2R P3; PDONR.TM. P2R-P3;
pDONR.TM. P4-P1R; pDONR.TM. P4-P1R; pDONR.TM./Zeo; pDONR.TM./Zeo;
pDONR.TM.201; pDONR.TM.201; pDONR.TM.207; pDONR.TM.207;
pDONR.TM.221; pDONR.TM.221; pDONR.TM.222; pDONR.TM.222;
pEF/myc/cyto; pEF/myc/mito; pEF/myc/nuc; pEFi/His A, B, and C;
pEF1/myc-His A, B, and C; pEF1/V5-HisA, B, and C; pEF4/myc-His A,
B, and C; pEF4N5-His A, B, and C; pEF5/FRT V5 D-TOPO;
pEF5/FRT/V5-DEST.TM.; pEF6/His A, B, and C; pEF6/myc-His A, B, and
C; pEF6/V5-His A, B, and C; pEF6A/5-His-TOPO; pEF-DEST51; pENTR
U6_verA_sz; pENTR/HirTO_verA_sz; pENTR-TEV/D-TOPO;
pENTR.TM./D-TOPO; pENTR.TM./D-TOPO; pENTR.TM./SD/D-TOPO;
pENTR.TM./SD/D-TOPO; pENTR.TM./TEV/D-TOPO; pENTR.TM.11;
pENTR.TM.1A; pENTR.TM.2B; pENTR.TM.3C; pENTR.TM.4; pET
SUMO_verA_sz; pET104.1-DEST_verA_sz; pET104-DEST; pET
160/GW/D-TOPO_verA sz pET160-DEST_verA_sz; pET161 D-TOPO; pET 161/G
W/D-TOPO_verA_sz; pET161-DEST_verA_sz; pEXPi-DEST pEXP2-DEST
pEXP3-DEST; pEXP3-DEST_vefA_sz; pEXP-AD502 pFastBac Dual pFastBad
pFastBacHTA pFastBacHT B pFaslBacHT C; pFLDa; pFliTrx; pFRT/lacZeo;
pFRT/lacZeo, pOG44, pcDNA5/FRT; pFRT/lacZeo2; pGAPZ A, B, and C;
pGAPZa A, B. and C; pGene/V5-His A, B, and C; pGeneBLAzer-TOPO;
pGeneBLAzer-TOPOverA sz; pGlow-TOPO; pH)1_-D2; pH1L-S1;
pHybLex/Zeo; pHyBLex/Zeo-MS2; pIB/His A, B, and C; pIBA/5-His Topo;
pIBA/5-His-DEST; plBA/5-His-TOPO; plZA/5-His; p!ZT/V5-His; pl_en!i4
BLOCK-iT-DEST; pLenti4/BLOCK-iT-DEST; pLenti4/TOA/5-DEST;
pLenti4/TOA/5-DEST_verA sz; pLenti4A/5-DEST; pLen114.''/5-DEST
verA_sz; pLenti6/BLOCK-tT-DEST; pl_entiS/BLOCK-iT-DEST_verA_sz;
pLenti6/UbCA/5-DEST; pLenti6/UbC/vSDEST_verA_sz; pLenli6A/5-DEST;
pLen!i6A/5-D-TOPO; plex; pMelBac A, B, and C; pMET A, B, and C;
pMETa A, B, C; pMIBA/5-His A, B, and C; pMIBA/5-His/CAT;
pMT/BioEase-DESTverAsz; pMT/BioEase.TM.-DEST; pMT/BioEase.TM.-DEST;
pMT/BiPA/5-His A, B, and C; pMTN5-His A, B, and C; pMTN5-His-TOPO;
pMT-DEST.TM. 48; pNMT; pNMT1-TOPO; pNMT41-TOPO; pNMT81-TOPO; pOG44;
pPIC3.5K; pPIC6 A, B, and C; pPIC6a A, B, and C; pPICZ A; pPICZ B;
pPICZ C; pPICZalpha A; pPICZalpha B; pPICZalpha C; pREP4; pRH3;
pRH5.sup.f; pRSET; pSCRE EN-iT/lacZ-DEST_verA_sz;
pSecTag/FRTA/5-His TOPO; pSecTag2 A, B, and C; pSecTag2/Hygro A, B,
and C; pSH18-34; pThioHis A, B, and C; pTracer-CMV/Bsd;
pTracer-CMV2; pTracer-EF A, B, and C; pTracer-EF/Bsd A, B, and C;
pTracer-SV40; pTrcHis A, B. and C; pTrcHis2 A, B, and C;
pTrcHis2-TOPO.RTM.; pTrcHis2-TOPO.RTM.; pTrcHis-TOPO.RTM.;
pT-Rex-DEST30; pT-Rex-DEST30; pT-Rex-DEST.TM. 31;
pT-REx.TM.-DEST31; pUB/BSD TOPO; pUB6A/5-His A, B, and C; pUC18;
pUC19; pUniN5 His TOPO; pVAX1; pVP22/myc-His TOPO.RTM.;
pVP22/myc-His2 TOPO.RTM.; pYC2.1-E; pYC2/CT; pYC2/Nt A, B. C;
pYC2-E; pYC6/CT; pYD1; pYES2; pYES2.1A/5-His-TOPO; pYES2/CT;
pYES2/NT; pYES2/NT A, B, & C; pYES3/CT; pYES6/CT; pYES-DEST.TM.
52; pYESTrp; pYESTrp2; pYESTrp3; pZeoSV2(-); pZeoSV2(+); pZErO-1;
pZErO-2.
[0082] Other terms used in the fields of recombinant nucleic acid
technology and molecular and cell biology as used herein will be
generally understood by one of ordinary skill in the applicable
arts.
[0083] The present invention, in some embodiments, provides
prescribed in silico workflows that empower a user with an easy way
to navigate through various subroutines within complex workflows
and to view, set, or change parameters within those subroutines. In
certain embodiments, for example, all of the subroutines that are
used in a workflow are provided to a user within one workspace,
which is a separately viewable area on a computer screen. A
subroutine within a workflow is a discrete task that can be
conveniently completed by a user and provides a convenient stopping
point in a workflow, typically in the laboratory implementation of
an in silico workflow. For example, where a nucleic acid is
amplified during a workflow, an amplification reaction is a
subroutine in certain exemplary embodiments of such workflow. The
prescribed workflows of the present invention provide a roadmap for
a user to help them easily visualize and navigate through the
various discrete tasks that are involved in completion of a
workflow.
[0084] The term "subroutine" as described above and used herein is
intended to describe a part of a complex biological/biotechnology
workflow, (i.e., a discrete task that can be conveniently completed
by a user and provides a convenient stopping point in a workflow,
typically in the laboratory implementation of an in silico
workflow), and may comprise a series of steps of one or more
sub-process of the subroutine. A plurality of subroutines that
complete a biological process, which may be performed/executed in a
sequential order, make up a biological/biotechnology workflow. The
subroutines of an in silico workflow of the present invention, are
typically laboratory subroutines, especially biological laboratory
subroutines, that is subroutines that are useful for, and sometimes
have a direct counterpart in, a biological workflow, such as a
biological workflow performed in a biological research laboratory.
Accordingly, the term subroutine as used in this specification is
different in meaning from the typical software code definition of
subroutine.
[0085] In an illustrative non-limiting example, a biological
workflow can be a cloning workflow, such as TOPO.RTM. Cloning,
which comprises at least three subroutines exemplified by: "1.
Amplify fragments to use in TOPO.RTM. reaction;" "2. Create
TOPO.RTM. clones;" and "3. Preview clones." Each of these three
subroutines further comprise one or more steps, for example, the
subroutine "1. Amplify fragments to use in TOPO reaction" comprises
one or more steps (also referred to as tasks) such as, "1) to
select the fragment(s) a user want to amplify by PCR for use in a
TOPO cloning reaction; 2) optionally to change the regions to
amplify in the selected molecules; 3) load/select PCR settings for
the amplification such as Tm, % GC of primer; primer length; type
of nucleotide to be amplified (DNA/RNA); etc)." Each step (or task)
allows a user to select/set one or more parameters associated with
that step, for example in the illustrative example to TOPO Cloning
workflow, in the subroutine of "1. Amplify fragments to use in TOPO
reaction" and in the step (task) of "1. To select the fragment(s) a
user wants to amplify" an example parameter may be "selecting a
fragment from a list of fragments provided by the software; or
typing in or importing in a fragment known to user that the user
desires to amplify. This exemplary cloning workflow and other
biotech workflows comprising one or more subroutines, each
subroutine comprising one or more steps or tasks, and each step
comprising selecting/inputting one or more parameters are described
in further detail in other parts of the disclosure (see also FIGS.
3-9).
[0086] Exemplary software and/or computer program products of the
disclosure may be used to perform a biological workflow in silico.
Embodiments also relate to in silico design of a method to produce
one or more biomolecules, chemical molecules, or commercial
biological products, such as biotechnology products (design of a
biological workflow). In silico workflows of the disclosure may be
used to make or produce one or more biomolecules, chemical
molecules, or commercial biological products such as biotechnology
products using one or more computer program products including data
collections of the disclosure such as but not limited to clone
collections and individual clones; vectors; hosts/modified hosts
(for example having modified/designed vectors) to make certain
biomolecules and/or biological products or have certain biological
properties; polypeptides, such as enzymes, antibodies, hormones;
nucleic acids such as various types of RNA, DNA, primers, probes;
libraries (e.g., cDNA libraries, genomic libraries, etc.); buffers,
growth media, purification systems, cell lines, chemical compounds,
fluorescent labels, functional assays, and variety of kits
including DNA and protein purification, amplification and
modification. These exemplary biomolecules, chemical molecules,
and/or commercial products are provided for example only and are
not intended to limit the present invention.
[0087] Those skilled in the art will recognize that the operations
of one or more embodiments of this disclosure may be implemented
using hardware, software, firmware, or combinations thereof, as
appropriate. For example, some processes can be carried out using
processors or other digital circuitry under the control of
software, firmware, or hard-wired logic. The term "logic" herein
refers to fixed hardware, programmable logic and/or an appropriate
combination thereof, as would be recognized by one skilled in the
art to carry out a recited function(s). Software and firmware can
be stored on computer-readable media. Some other processes can be
implemented using analog circuitry, as is well known to one of
ordinary skill in the art. Additionally, memory or other storage,
as well as communication components, may be employed in embodiments
of the disclosure.
[0088] FIG. 1 illustrates an example of a computing system and
client/server environments, database servers, and networks and
their interconnections. FIG. 1 provides an exemplary client/server
system 100 showing the general architecture for performing methods
provided herein. The general architecture includes server
functions, including design of biomolecules such as RNAi, and other
scripts which are run on a server computer 102 and that can access
databases on the server (104, 106, and 108), and web pages 110 that
are delivered to the client. Server computer 102 may be connected
to the internet via an internet service provider (ISP). Similarly,
client computers may be connected to the internet via an ISP
connection. Client/server system 100 may include a user list
database 104 for storing users of the system. In some embodiments,
users may need to login to the system to access information.
Client/server system 100 may also include user data database 106
that may include data stored associated with the plurality of users
of the system. For example, a customized workflow file may be
stored in the user data database 106. Further, client/server system
100 may include a company database 106 and/or a public access
database 106 (such as GenBank that a user may use to obtain data
from) or a commercial database 106 including databases stored on a
cloud that may include product information that a user may use to
produce a biomolecule, chemical, and/or commercial product such as
a biotechnology product. Further, client/server system 100 may
include a company database 108 that may include product information
that a user may use to produce a biomolecule, chemical, and/or
commercial product such as a biotechnology product. A company may
update available products periodically to remove products that may
not be available anymore or add new products, for example.
Furthermore, other predetermined workflow files may be uploaded to
company database 108 to be downloaded or provided to users.
Typically, the server computer is maintained by a provider of
biological products and of the computer products provided herein,
and the client computer is a computer of the customer. In various
embodiments, there is a plurality of client computers in
communication with server 102. Client computers may display user
interfaces such as webpages 110 or molecule viewers 112 according
to various embodiments described herein, for example.
[0089] In some embodiments, server 102 or another server in
communication with client computers may store user data such that a
user may download data, including workflows, from the server.
Furthermore, a user may store data that may be accessed by other
users of the client/server system 100. For example, according to
some embodiments described herein, a prescribed workflow may be
shared with another user or a group of users.
[0090] As mentioned above, according to various embodiments, user
data may be stored in the user data database 106. User data may
include user feedback on a workflow or a biomolecule, chemical
molecule, and/or commercial product that results from carrying out
a workflow of the invention, for example. In various embodiments,
the user data may be further analyzed to generate personalized
recommendations for a user, such as commonly used parameters by the
user or offer recommendations of commercial products the user may
want to purchase. The aggregate of user data may further be
analyzed, according to various embodiments, to generate statistics
on prescribed workflows or biomolecules, chemical molecules, and/or
commercial products that result from carrying out a workflow of the
invention.
[0091] In another aspect of the invention, a documented Application
Programming Interface (API) is provided to a customer that is
associated with an in silico design method, an in silico workflow
method, and a computer program product. API further can provide
product ordering options to a customer such that a customer can
route orders through that customer's computer system, such as a
business-to-business system.
[0092] FIG. 2 is a block diagram that illustrates a computer system
700 that may be employed to carry out processing functionality,
according to various embodiments. Computing system 700 can include
one or more processors, such as a processor 704. Processor 704 can
be implemented using a general or special purpose processing engine
such as, for example, a microprocessor, controller or other control
logic. In this example, processor 704 is connected to a bus 702 or
other communication medium.
[0093] Further, it should be appreciated that a computing system
700 of FIG. 2 may be embodied in any of a number of forms, such as
a rack-mounted computer, mainframe, supercomputer, server, client,
a desktop computer, a laptop computer, a tablet computer, hand-held
computing device (e.g., PDA, cell phone, smart phone, palmtop,
etc.), cluster grid, netbook, embedded systems, or any other type
of special or general purpose computing device as may be desirable
or appropriate for a given application or environment.
Additionally, a computing system 700 can include a conventional
network system including a client/server environment and one or
more database servers, or integration with LIS/LIMS infrastructure.
A number of conventional network systems, including a local area
network (LAN) or a wide area network (WAN), and including wireless
and/or wired components, are known in the art. Additionally,
client/server environments, database servers, and networks as
illustrated in the example in FIG. 1 are well documented in the
art.
[0094] Computing system 700 may include bus 702 or other
communication mechanism for communicating information, and
processor 704 coupled with bus 702 for processing information.
[0095] Computing system 700 also includes a memory 706, which can
be a random access memory (RAM) or other dynamic memory, coupled to
bus 702 for storing instructions to be executed by processor 704.
Memory 706 also may be used for storing temporary variables or
other intermediate information during execution of instructions to
be executed by processor 704. Computing system 700 further includes
a read only memory (ROM) 708 or other static storage device coupled
to bus 702 for storing static information and instructions for
processor 704.
[0096] Computing system 700 may also include a storage device 710,
such as a magnetic disk, optical disk, or solid state drive (SSD)
is provided and coupled to bus 702 for storing information and
instructions. Storage device 710 may include a media drive and a
removable storage interface. A media drive may include a drive or
other mechanism to support fixed or removable storage media, such
as a hard disk drive, a floppy disk drive, a magnetic tape drive,
an optical disk drive, a CD or DVD drive (R or RW), flash drive, or
other removable or fixed media drive. As these examples illustrate,
the storage media may include a computer-readable storage medium
having stored therein particular computer software, instructions,
or data.
[0097] In alternative embodiments, storage device 710 may include
other similar instrumentalities for allowing computer programs or
other instructions or data to be loaded into computing system 700.
Such instrumentalities may include, for example, a removable
storage unit and an interface, such as a program cartridge and
cartridge interface, a removable memory (for example, a flash
memory or other removable memory module) and memory slot, and other
removable storage units and interfaces that allow software and data
to be transferred from the storage device 710 to computing system
700.
[0098] Computing system 700 can also include a communications
interface 718. Communications interface 718 can be used to allow
software and data to be transferred between computing system 700
and external devices. Examples of communications interface 718 can
include a modem, a network interface (such as an Ethernet or other
NIC card), a communications port (such as for example, a USB port,
a RS-232C serial port), a PCMCIA slot and card, Bluetooth, etc.
Software and data transferred via communications interface 718 are
in the form of signals which can be electronic, electromagnetic,
optical or other signals capable of being received by
communications interface 718. These signals may be transmitted and
received by communications interface 718 via a channel such as a
wireless medium, wire or cable, fiber optics, or other
communications medium. Some examples of a channel include a phone
line, a cellular phone link, an RF link, a network interface, a
local or wide area network, and other communications channels.
[0099] Computing system 700 may be coupled via bus 702 to a display
712, such as a cathode ray tube (CRT) or liquid crystal display
(LCD), for displaying information to a computer user. An input
device 714, including alphanumeric and other keys, is coupled to
bus 702 for communicating information and command selections to
processor 704, for example. An input device may also be a display,
such as an LCD display, configured with touchscreen input
capabilities. Another type of user input device is cursor control
716, such as a mouse, a trackball or cursor direction keys for
communicating direction information and command selections to
processor 704 and for controlling cursor movement on display 712.
This input device typically has two degrees of freedom in two axes,
a first axis (e.g., x) and a second axis (e.g., y), that allows the
device to specify positions in a plane. A computing system 700
provides data processing and provides a level of confidence for
such data. Consistent with certain implementations of embodiments
of the present teachings, data processing and confidence values are
provided by computing system 700 in response to processor 704
executing one or more sequences of one or more instructions
contained in memory 706. Such instructions may be read into memory
706 from another computer-readable medium, such as storage device
710. Execution of the sequences of instructions contained in memory
706 causes processor 704 to perform the process states described
herein. Alternatively hard-wired circuitry may be used in place of
or in combination with software instructions to implement
embodiments of the present teachings. Thus implementations of
embodiments of the present teachings are not limited to any
specific combination of hardware circuitry and software.
[0100] The term "computer-readable medium" and "computer program
product" as used herein generally refers to any media that is
involved in providing one or more sequences or one or more
instructions to processor 704 for execution. Such instructions,
generally referred to as "computer program code" (which may be
grouped in the form of computer programs or other groupings), when
executed, enable the computing system 700 to perform features or
functions of embodiments of the present disclosure. These and other
forms of computer-readable media may take many forms, including but
not limited to, non-volatile media, volatile media, and
transmission media. Non-volatile media includes, for example, solid
state, optical or magnetic disks, such as storage device 710.
Volatile media includes dynamic memory, such as memory 706.
Transmission media includes coaxial cables, copper wire, and fiber
optics, including the wires that comprise bus 702.
[0101] Common forms of computer-readable media include, for
example, a floppy disk, a flexible disk, hard disk, magnetic tape,
or any other magnetic medium, a CD-ROM, any other optical medium,
punch cards, paper tape, any other physical medium with patterns of
holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip
or cartridge, a carrier wave as described hereinafter, or any other
medium from which a computer can read.
[0102] Various forms of computer readable media may be involved in
carrying one or more sequences of one or more instructions to
processor 704 for execution. For example, the instructions may
initially be carried on magnetic disk of a remote computer. The
remote computer can load the instructions into its dynamic memory
and send the instructions over a telephone line using a modem. A
modem local to computing system 700 can receive the data on the
telephone line and use an infra-red transmitter to convert the data
to an infra-red signal. An infra-red detector coupled to bus 702
can receive the data carried in the infra-red signal and place the
data on bus 702. Bus 702 carries the data to memory 706, from which
processor 704 retrieves and executes the instructions. The
instructions received by memory 706 may optionally be stored on
storage device 710 either before or after execution by processor
704. In some embodiments wireless internet connectivity can be used
to access and receive data from by remote computer.
[0103] FIG. 10 is a block diagram that illustrates a typical
Internet network configuration wherein a number of client machines
1402 possibly in a remote local office, are shown connected to a
gateway/hub/tunnel-server/etc. 1410 which is itself connected to
the internet 1408 via some internet service provider (ISP)
connection 1410. Also shown are other possible clients 1412
similarly connected to the internet 1408 via an ISP connection
1414, with these units communicating to possibly a central lab or
office, for example, via an ISP connection 1416 to a
gateway/tunnel-server 1418 which is connected 1420 to various
enterprise application servers 1422 which could be connected
through another hub/router 1426 to various local clients 1430. Any
of these servers 1422 could function as a development server for
the analysis of potential content management and delivery design
solutions as described in the present disclosure, as more fully
described below.
[0104] It will be appreciated that, for clarity purposes, the above
description has described embodiments of the disclosure with
reference to different functional units and processors. However, it
will be apparent that any suitable distribution of functionality
between different functional units, processors or domains may be
used without detracting from various embodiments of this
disclosure. For example, functionality illustrated to be performed
by separate processors or controllers may be performed by the same
processor or controller. Hence, references to specific functional
units are only to be seen as references to suitable means for
providing the described functionality, rather than indicative of a
strict logical or physical structure or organization.
[0105] In some embodiments of the present disclosure, in silico
methods are described that may be performed (executed), by a user,
to obtain a biotechnology product comprising one or more steps
(e.g., a workflow or in some embodiments multiple workflows to
obtain a pipeline of workflows) that may be accessible and
controllable by the user via a Graphical User Interface (GUI) that
is visible on Display 712. A user may enter data (e.g. external
data) and/or select options provided in the GUI using Input Device
714 and/or Cursor Control 716. In some embodiments, components of
computer system 700 convert input data provided by a user into a
computer readable format to one or more computer system components
(such as a memory, a database, a processor etc.) to enable
interpretation of input data received from a user and to initiate
controller instructions to conduct one or more steps of the in
silico method.
[0106] In some embodiments, user input data may also be used for
report generation of the particular in silico method being
performed. In some embodiments, components of computer system 700,
such as Display 712, may also receive data from one or more
processors/sensors/detectors following performing one or more steps
of an in silico method that are then converted into a user
understood format to enable a user to monitor progress of the
workflow steps and/or to obtain additional input from a user to
determine the next course/step of the workflow in the in silico
method. Input of data from a user or translation of data received
from various devices within computer system 700 may be mediated by
components of a software (or computer program) of the disclosure
(not expressly depicted in FIG. 6) which comprises comprising a
computer readable medium comprising computer readable instructions,
which, when executed by the computer system, are configured to
display on Display 712 (screen, LCD).
[0107] A software (or computer program) of the disclosure may be
operable to receive user instructions, either in the form of user
input into a set parameter fields, e.g., in a GUI, or in the form
of pre-programmed instructions such as but not limited to
pre-programmed instructions for performing a variety of different
specific operations and/or for analyzing various parameters and/or
for analyzing one or more data components. A software of the
disclosure, in some embodiments, may be operable to convert
pre-programmed instructions to appropriate computer language for
instructing operation of system 700 to carry out a desired
operation. A software of the disclosure, in some embodiments, may
be operable to convert data signals or parameters received into
appropriate computer language that may then be analyzed by a
processor in computer and/or converted into user viewable format
for a user to review or analyze.
[0108] In some embodiments, a software of the disclosure may
comprise functional specifications as well as graphical user
interface (GUI) specifications. GUI specifications enable user
mediated methods. Exemplary GUI's of the present disclosure may
comprise some general GUI specifications. In some embodiments,
general GUI specifications may comprise all screens, with the
exception of pop-up screens, being 800 pixels wide and 480 pixels
high.
[0109] Other general GUI specifications may include without
limitation, the availability of a Home button in all menu screens
where Home button allows a user to navigate to a Main Menu; the
availability of Breadcrumbs or a Breadcrumb Trail in all menu
screens (breadcrumbs may be abbreviated when they are too long for
display); the availability of Time and Date in all menu screens;
the availability of a Back button in all menu screens where a Back
button allows a user to navigate to a previous screen; the
availability of a Save button in screens where a user can change
and save one or more fields. Breadcrumbs refer to a navigation aid
used in a user interface to show the path that a user has taken to
arrive at a screen.
[0110] In some embodiments, in a screen where a Save button is
available, a Back button may allow a user to either save or cancel
a change, if any, before navigating to previous screen. In some
embodiments, in a screen where a Save button is available, a Home
button allows a user to either save or cancel a change, if any,
before navigating to a Home screen. General GUI specifications also
include the availability of a Keypad in screens where a user needs
to enter an alpha-numeric string or special character keys. Some
examples of GUI's of the disclosure are described later in this
application.
[0111] Exemplary software and/or computer program products of the
invention can be used to perform in silico design of a method to
produce one or more biomolecules, chemical molecules, or
biotechnology products (design of a biological workflow). In silico
designing to make or produce one or more biomolecules, chemical
molecules, or biotechnology products using one or more computer
program products of the invention can include production of
biomolecules, chemical molecules, or biotechnology products such as
but not limited to clone collections and individual clones;
vectors; hosts/modified hosts (for example having modified/designed
vectors) to make certain biomolecules, chemical molecules, or
biotechnology products or have certain biological properties;
polypeptides, such as enzymes, antibodies, hormones; nucleic acids
such as various types of RNA, DNA, primers, probes; libraries
(e.g., cDNA libraries, genomic libraries, etc.); buffers, growth
media, purification systems, cell lines, chemical compounds,
fluorescent labels, functional assays, and variety of kits
including DNA and protein purification, amplification and
modification. Further, these exemplary products are provided for
example only and are not intended to limit the present
invention.
[0112] One or more methods of the disclosure can be performed in
silico using a computer system comprising a non-transitory computer
readable storage medium encoded with instructions, comprising
computer readable instructions (such as a computer program), which,
are executable by a processor of the computer system. In some
embodiments, instructions of a computer readable storage medium of
the disclosure may comprise instructions to display on a display
screen series of steps that may be performed to obtain a
biotechnology product. In some embodiments, the displayed series of
steps comprise all the steps that must be performed by a user to
obtain a biotechnology product and may be referred to in some
embodiments as "prescribed steps" or a "prescribed workflow" or "a
subroutine of a biological workflow." A series of "prescribed
steps" or "subroutines" may be displayed on a GUI navigation panel
(or display pane) where selecting a subroutine (button on GUI)
highlights it and takes a user to another navigation panel or GUI
screen (or display pane) which provides a list of steps (tasks)
that comprise the selected subroutine. Each individual subroutine
of a biological workflow generally further comprise a series of
steps (sub-steps or tasks) each of which may be executed
independently to obtain intermediate products of the biotechnology
product. Accordingly, in some embodiments, instructions of a
computer readable storage medium of the disclosure may comprise
instructions to display on a display screen (such as a second
display screen or a second display pane), steps (tasks or
sub-steps) of the "subroutine" or "prescribed step" selected by a
user using a GUI button in a first display screen or display
pane.
[0113] Using a GUI a user may customize one or more of these steps
(tasks or substeps) by providing user inputs. In some embodiments,
user inputs may comprise customized inputs that may be user
designed (generated by a user, imported by a user or modified from
default parameters by a user). In some embodiments, user inputs may
be selected from a set of default inputs that are comprised/stored
in the computer program (for example a database having default
alternative parameters/values that may be available for the user to
select (for example, in the form of a drop down menu)).
[0114] In silico methods of the disclosure, in some embodiments,
may comprise a user navigating through "prescribed steps" or
"subroutines" in a sequential fashion (navigation in ordered
steps). In other embodiments, a user may navigate through
"prescribed steps" or "subroutines" in no particular sequence and
may even go back and forth to input data into various steps out of
order (navigation in random steps) prior to performing (executing)
the entire prescribed step series.
[0115] In some embodiments, a computer-readable storage medium
encoded with instructions, executable by a processor, the
instructions comprising instructions for: 1) executing individual
steps of a "prescribed series of steps" (also called "subroutine");
and 2) instructions for viewing intermediate products obtained by
one or more of the steps of the "prescribed steps" thereby allowing
a user to view an intermediate biotechnology product and determine
if the method step(s) need to be modified to arrive at a final
biotechnology product.
[0116] Accordingly, the present disclosure provides a user with a
tool to review progress of intermediate steps of a biotechnology
process by viewing intermediate biotechnology products and
providing the ability to change one or more conditions, parameters
and/or criteria associated with that intermediate step by
inputting/selecting another parameter and/or criteria if the
intermediate biotechnology product was not found to be satisfactory
or optimum, thereby allowing the user to design a better method for
making the final biotechnology product. In some embodiments, a user
may be able to determine what parameters to input into a prescribed
method based on ability to review the outcome (intermediate product
and/or final product) in silico.
[0117] A user may conveniently navigate a GUI of prescribed steps
in any random or ordered fashion and input a parameter (such as by
determining what parameter to input for the best possible outcome
as described above). Once a user has made selections and/or input
parameters for all steps of a prescribed method, the entire method
may be executed in silico and the final product may be viewed.
[0118] In some embodiments of the disclosure, a computer-readable
storage medium encoded with instructions, executable by a
processor, may comprise instructions for: 3) storing each user
selected/input parameter associated with each step/sub-step of a
biotech method in a memory and 4) instructions for allowing a user
to retrieve the stored parameters. Accordingly a user may store and
retrieve a log of one or more user-defined method (user defined
workflow) comprising information that was input/selected by a user,
in the form of a electronic lab note book, thereby accurately
capturing all changes made/all parameters input by user in a
method. This allows for accurate reproduction and tracking of
changes made to a workflow for obtaining a biotechnology product.
In some embodiments, a user-defined method (user defined workflow)
that is stored may be then converted into user viewable format for
display, and/or copying and/or sending to the same or different
user in various human readable formats (email, html etc). In some
embodiments, optimized methods designed by the in silico methods
described herein can be shared by a plurality of users.
[0119] Methods of the disclosure can further include performing
laboratory steps (corresponding to the in silico steps) to confirm
and possibly expand the determinations made using the in silico
methods to produce a biotechnology product that is optimal (in
quality and/or quantity (yield)) and/or arrive at a biotechnology
method with optimal efficiency for producing a biotech product.
[0120] In some embodiments, a biotechnology process of the
disclosure, may be a computational biology process(es).
Biotechnology processes and their analysis is often carried out in
multistep processes that can be generalized steps, customized steps
and/or a combination of generalized and customized steps.
[0121] In some embodiments, steps for a biotechnology process,
according to the present teachings may comprise an ordered sequence
of steps (wherein step 1 must be followed by step 2). In some
embodiments, steps of a biotechnology process may comprise an
unordered/random sequence of steps (wherein steps 1, 2, 3, . . .
may be performed in no specific order or pattern). In some
embodiments of an in silico method, for an unordered series of
method steps a final confirmation step comprising a user input
provided has to be provided by the user before the final step of
the process can be executed.
[0122] FIG. 3 depicts an exemplary in silico method 800 according
to one embodiment of the present invention. In silico method 800
includes a plurality of set workflow subroutines in a computer
readable format that can include subroutines for a biotechnology
process depicted in FIG. 3 as Subroutine 10, Subroutine 20,
Subroutine 30 . . . and so on. FIG. 3 is merely an exemplary method
and the skilled artisan, in light of this disclosure, will realize
that the actual number of subroutines can vary from at least about
2 subroutines to many (e.g. 2-10, 2-20, 2-30, 2-n (where n may be
any number of subroutines from 3-100, 3-1000 and so on)). Each set
subroutine 10, 20, 30, 40, 50, 60 . . . etc. can include a single
step or task, or optionally can include more than 1 step or task
(e.g. Steps A, B, C . . . shown as Step 20A, 20B, 20C for
Subroutine 20; and as 30A, 30A1, 30A2 for Subroutine 30), also in a
computer readable format, and each step can further include
additional optional customizable steps or tasks shown in FIG. 3 as
Steps A1, A2, B1, B2, C1 C2 . . . (e.g. shown as Step 20A1, 20A2
for Step 20A; and Step 20B1, 20B2 for Step 20B of Subroutine 20;
and as Step 30A1, 30A2 for Step 30A; and Step 30B1, 30B2 for Step
30B of Subroutine 30,). Each of the optional/customizable steps or
tasks can have one or more optional parameters (options) that can
be viewed, reviewed, set or customized by a user for example see 1,
2, 3, 1a, 2a 1b, 2b, 1', 2' . . . . In some embodiments, an in
silico method of the invention includes selection by a user of at
least one parameter each for each optional/customizable step of the
biotechnological process using a graphical user interface (GUI) to
select the at least one parameter for each optional/customizable
step. In certain embodiments, every step and every parameter of the
subroutines of a workflow are available to a user to view, and
optionally edit. Bioinformatics programs typically hide some of
these parameters and/or steps from users, which causes user
frustration and inefficiency especially when the result of an in
silico designed experiment is not the expected result for a
user.
[0123] An exemplary in silico method of the disclosure illustrated
generally in FIG. 3 can be carried out (performed) by generating at
least one method file in a computer system, the method file
comprising computer readable instructions for a plurality
subroutines (10, 20, 30 . . . ) of customizable steps (A, B, C)
each of which may have one or more parameters that may be viewed,
selected, changed or inputted; and performing the biotechnological
process in silico comprising executing the at least one method file
comprising computer readable instructions by the computer system to
obtain at least one biotechnology product.
[0124] In some embodiments, at least one customizable/optional
parameter is selected from a default parameter, wherein the default
parameter is stored in a component of the computer system (such as
storage, database etc.).
[0125] Accordingly, in some embodiments, the present disclosure
provides a non-transitory computer-readable storage medium encoded
with instructions, executable by a processor, the instructions
comprising instructions for providing a method for performing a
biological workflow in silico comprising: presenting to a user a
plurality of subroutines that comprise the biological workflow
listed in a sequential order, wherein at least two subroutines of
the plurality of subroutines comprise two steps; providing the user
ability to navigate to any subroutine of the plurality of
sequential subroutines, to select a subroutine and view, set,
select, or change one or more parameters associated with a step of
the selected subroutine; providing an option to display to the
user, one or more biomolecules resulting from execution of one or
more of the plurality of subroutines; and providing an option to
the user to navigate to a prior subroutine and change a parameter
of a step of the prior subroutine, if the user is not satisfied
with the one or more displayed biomolecules.
[0126] In some embodiments, a method of the disclosure may further
comprise, providing an indication to the user of a current
subroutine of the method for performing a biological workflow for
the user to review. An indication may be provided by highlighting
in a GUI the current subroutine. A GUI type display may also be
used to display to a user the number of steps in a selected
subroutine. In some embodiments, multiple display panes in a GUI
may be used, one display pane to show the subroutine step and
another display pane to show the steps in that subroutine.
[0127] Some embodiments also provide an indication of a current
step by highlighting for example a step or task within a subroutine
to a user to which a user has navigated.
[0128] In some embodiments, a method of the disclosure may further
comprise, receiving at least one selection desired by the user,
from the user, for one or more parameters associated with at least
one step of the plurality of subroutines of the method for
performing a biological workflow; and providing to the user an
option for storing in a memory a user defined workflow comprising
settings selected by the user for parameters used in the biological
workflow. A user defined workflow may also be described herein as a
user defined method of a biological workflow.
[0129] In some embodiments, a method of the disclosure may further
comprise providing to the user the ability to rate or comment on
the user defined workflow and storing the rating associated with
the user defined workflow in the memory.
[0130] In some embodiments, a method of the disclosure may further
comprise providing to a user, preset settings for parameters based
on user defined workflows with the highest ratings or the most
positive comments. In some embodiments, a method of the disclosure
may further comprise instructions for ability for a plurality of
users to provide feedback about parameters selected for optimally
performing the biological workflow; and storing feedback received
from the plurality of users in a memory. In such embodiments, the
software is able to "learn" from its experience based on user input
and rating. Software of the disclosure may in some embodiments have
neural networks and artificial intelligence capability.
[0131] In some exemplary embodiments, analysis of feedback provided
by a community of users may be used to determine which parameters
may be better suited for a particular step of a subroutine. For
example, in a non limiting example of a biological workflow for
designing a recombinant biomolecule some example optional
parameters that may be selected by users in a step of a subroutine
may be selection of a vector from several possible vector choices
and selection of one open reading frame. A plurality of vectors and
open reading frame choices may be stored in a memory (database) and
presented as optional parameters to users (for example in a drop
box format in a GUI), thereby allowing the user to design several
possible recombinant biomolecules by selecting different
combinations of vectors and open reading frames. A user could thus
determine, in silico (and also additionally in the lab if desired),
which combination would lead to the most optimum recombinant
biomolecule and/or therefore determine the most optimum
user-defined workflow for making the optimum biomolecule. If
multiple users provide comments, rating or feedback that certain
vectors are consistently not optimal; and/or a certain open reading
frame are consistently not optimal across multiple users; and/or
that a certain reading frame in combination with certain vectors do
not provide an optimal recombinant biomolecule as desired, the
software is designed to "learn" from this input and provide to a
future user the information that certain vectors and ORF's may not
be optimal for use in a workflow for making the recombinant
biomolecule. On the contrary, if previous user input indicates that
certain vectors are good, the software may display to a future user
a list of vectors (and ORF's) that worked well in workflows for
other users.
[0132] In some embodiments, the ability of the software to store
user feedback and user workflows may allow a provider to make a kit
selecting certain parameters (such as specific vectors, and ORF's
in the example above), and/or sell or market certain products for
use in a corresponding laboratory workflow.
[0133] In another exemplary embodiment, a biological workflow may
comprise restriction cloning and once a user uploads a DNA fragment
to be cloned the software may display options of Restriction
Endonucleases (RE) that may be used to cut the DNA fragment from a
list of possible enzymes based on the sequence. However, since some
RE's are more expensive that others, users will preferentially
never select the more expensive RE's from the list if another
choice is available. A software of the disclosure is able to learn
from repeated selections (and or comments, feedback) from multiple
users and be able to suggest to a future user a less expensive
choice of a RE, thereby able to generate a cost effective
biological workflow for a user.
[0134] Accordingly, in some embodiments a non-transitory
computer-readable storage medium of the present disclosure, encoded
with instructions and executable by a processor may additionally
comprise instructions for generating recommendations of parameters
based on feedback received from the plurality of users and
presenting generated recommendations to future users. In some
embodiments, recommendations may be subroutine specific
recommendations. In some embodiments, recommendations may be step
specific recommendations.
[0135] In some embodiments, a method of the disclosure may further
comprise providing the user the ability to navigate to any
subroutine of the plurality of sequential subroutines to select a
subroutine and view, set, or change one or more parameters
associated with a step of the selected subroutine, wherein the
parameter being viewed, set or changed is a parameter that results
from ambiguous user data.
[0136] In some illustrative non-limiting exemplary embodiments,
ambiguous user data may be generated for example by users during
sequencing old DNA. Old DNA may include mummified DNA and/or
fossilized DNA wherein the quantity of DNA is limited and the
quality of DNA is poor due to the nature of the source of DNA.
Typically workflows such as ContigExpress (explained and
illustrated in detail in sections below and FIGS. 6 and 9) are used
to build contig's of fragments sequenced from such old DNA and
comprise several "subroutines" including "Subroutine 1=View
Fragments"; "Subroutine 2=Trim ends;" "Subroutine 3=Trim vector
contaminations;" "Subroutine 4=Assemble contig;" and "Subroutine
5=View Contig." Prior to the subroutines 1 of "viewing fragments" a
user will typically "sequence DNA fragments" and upload them into a
molecular viewed of the software. Due to the poor DNA quality there
is a lot of "fuzziness" at ends of fragments, which is due to the
inability of a DNA sequencing machine to determine accurately the
intensity of signal of nucleotides (for example, a capillary
electrophoresis machine is unable to detect clearly is a peak is an
"A" or "T").
[0137] In this example, while performing the "subroutine 1 of View
Fragments" typically all bioinformatics software automatically trim
off the "fuzzy" end fragments, where the sequence is not clearly
readable. Typically, bioinformatics software does this inherent
trimming of "fuzzy" ends and provides no indication to a user of
the fact that such trimming was done. Typically this frustrates and
confuses users as they do not see the expected length of fragment
in the display and have no way of knowing what happened (no way of
knowing that bioinformatics software typically trims fuzzy ends).
The present software, in contrast to existing software, keeps a log
(electronic log) of all the steps it executed and a viewer, is
confused, can navigate and look at the electronic log and see a
Table that will show the nucleotides and positions that were
trimmed and a note of why they were trimmed in the example of where
a fragment viewed does not have an expected length (for example).
The present software also provides in a subroutine options to view,
select, change, steps/parameters to not automatically remove such
ambiguous user data. For example, in this illustrative example, the
present software has the ability to allow a user to select a
step/parameter that does not trim the fuzzy lengths off.
[0138] In some embodiments, a method of the disclosure may further
comprise providing the user the ability to navigate to any
subroutine of the plurality of sequential subroutines to select a
subroutine and view, set, or change one or more parameters
associated with a step of the selected subroutine, wherein ever
parameter in a workflow that is set based on ambiguous user data
can be viewed, set, or changed by the user.
[0139] In some embodiments, a method of the disclosure may further
comprise providing the user the ability to navigate to any
subroutine of the plurality of sequential subroutines to select a
subroutine and view, set, or change one or more parameters
associated with a step of the selected subroutine, wherein ever
parameter in a workflow that is set based on ambiguous user data is
highlighted to a user such that the parameter can be viewed, set,
or changed by the user.
[0140] In some embodiments, the non-transitory computer-readable
storage medium also provides to a user the ability to save a log of
all selected parameters for each step of the user defined method;
and also optionally receive and store from the user at least one
comment about the user defined method. Therefore, in some
embodiments, an electronic lab notebook function is provided which
allows a user to carefully look back at each and every selection
and analyze the outcome of a biological workflow.
[0141] In some embodiments, the software of the present disclosure
also has the capability for storing one or more external files
uploaded by the user; and providing to the user or to additional
users ability to view the one or more external files uploaded. This
may be helpful when multiple users run an experiment. For example,
in a cloning workflow, if 5 inserts are desired to be cloned into 5
vectors, an excel file of all possible combinations of vector and
inserts may be made and multiple users can enter data an update the
excel sheet as a progressive log book of the experiment. Various
versions of a file may be uploaded and accessed.
[0142] In some embodiments, viewing, selecting, setting, or
changing one or more parameters associated with a step of the
selected subroutine by the user comprises using a graphical user
interface (GUI).
[0143] In some embodiments, the one or more parameters viewed,
selected, set or changed by the user are from one or more default
parameters, wherein default parameters are pre-determined
parameters stored in the computer-readable storage medium.
[0144] In some embodiments, the one or more parameters viewed,
selected, set or changed by the user comprise at least one user
input parameter.
[0145] In some embodiments, the user input parameter is a modified
default parameter, a parameter input by user, or a parameter
imported by the user into the computer system, wherein a default
parameter is a pre-determined parameter stored in a component of
the computer-readable storage medium.
[0146] In some embodiments, the one or more parameters viewed,
selected, set or changed by the user comprise a combination of one
or more default parameters and one or more user defined parameters,
wherein default parameters are pre-determined parameters stored in
a component of the computer-readable storage medium and wherein the
user input parameter is a modified default parameter, a parameter
input by user, or a parameter imported by the user into the
computer system.
[0147] In some embodiments, providing the user ability to navigate
to any subroutine of the plurality of subroutines is by a graphical
user interface (GUI) which comprises displaying on a first display
screen pane all the subroutines of a sequential subroutine
comprising the biological workflow and, following selection by the
user of any one subroutine, displaying on a second display screen,
one or more steps associated with the selected subroutine.
[0148] In some embodiments, providing the user ability to navigate
to any step of a subroutine is by a graphical user interface (GUI)
which comprises displaying on a first display screen pane the
subroutine and displaying on a second display screen, one or more
steps associated with the selected subroutine.
[0149] In some embodiments, providing to the user an option for
saving on the computer readable storage medium a user defined
method comprising at least one selection desired by the user
comprises: receiving selections by the user of one or more
parameters for each step of the plurality of subroutines of the
biological workflow and saving the selections as a selected
plurality of steps, wherein the saved selected plurality of steps
comprise the user-defined method; and storing a user defined name
for the user-defined method.
[0150] In some embodiments, providing to the user an option for
saving on the computer readable storage medium a user defined
method comprising at least one selection desired by the user
comprises: a) displaying on a display screen pane a prescribed
plurality of subroutines in a sequential order of the biological
workflow, wherein the prescribed plurality of subroutines are
comprised in a computer readable format; b) navigation by a user
using a graphical user interface (GUI) on the display screen of
each subroutine of the prescribed plurality of subroutines of the
biological workflow; c) selection by the user of one of the
subroutines; d) navigation by a user using a GUI on the display
screen of each step of a selected subroutine; e) selection by the
user of one or more parameters in each step of a selected
subroutine to obtain a modified plurality of steps; f) storing the
modified plurality of steps by the user, wherein the stored
modified plurality of steps comprise the user defined subroutine;
g) repeating steps b)-f) till all the plurality of sub routines are
stored as user defined subroutines; and h) saving and executing the
plurality of user defined subroutines to perform a user defined
biological workflow.
[0151] In some embodiments, navigation by a user of any subroutine
or step of the workflow is in a sequential order of the steps or
the subroutine. In some embodiments, navigation by a user of any
subroutine or step of the workflow is in a non-sequential order of
the steps or the subroutine.
[0152] In some embodiments, an in silico workflow may further
comprise executing a first user defined workflow comprising
executing in silico all the steps of a first user defined workflow
in sequential order; viewing a first biomolecule obtained by
executing the first user defined workflow in silico; generation of
at least a second user defined workflow, comprising changing the
selection of at least one parameter to have a different value
relative to the same parameter that was selected in the first user
defined workflow; executing in silico the at least second user
defined workflow to obtain a second biomolecule; viewing the second
biotechnology product in silico; and comparing the first
biomolecule with the second biomolecule, thereby allowing a user to
determine if the first user defined workflow or the second user
defined method is better. In this embodiment, the ability to
accurately determine the parameters by means to the stored
workflows in a memory, allow changing one parameter in relation to
another.
[0153] Exemplary non-limiting biological workflow include a cloning
method, a recombination method, a ligation method, a vector
designing method, a method for synthesis of a nucleic acid, a
primer design method, a method for synthesis of a polypeptide,
method for analysis of a cloned molecule, method of protein
analysis, method for making a modified host.
[0154] In some embodiments, a non-transitory computer-readable
storage medium of the disclosure, encoded with instructions,
executable by a processor, the instructions comprising instructions
for: providing a pipeline of a method for performing a biological
workflow for display to a user, the pipeline of a method comprising
a plurality of methods, each method generating at least one
biomolecule that may be used in the next method to produce another
biomolecule, wherein subroutines of each of the plurality of
methods are listed in a sequential order; and executing the
pipeline of the method.
[0155] In some embodiments, generation of the at least one method
file may comprise selection by a user of at least one default
parameter or selection of at least one user input parameter for
each step of the biotechnological process, wherein the at least one
default parameter is stored in a component of the computer system
and wherein the at least one user input parameter is a modified
default parameter, a parameter input by user, or a parameter
imported by the user into the computer system.
[0156] In some embodiments, the generation of the at least one
method file may comprises: displaying on a display screen at least
two or more sequential subroutines (subroutines 10, 20, 30 . . . as
in FIG. 3) of a prescribed workflow of a biotechnological process,
wherein the two or more steps are comprised in a computer readable
format in the computer system; navigation by a user using a
graphical user interface (GUI) on the display screen of each of the
two or more subroutines (go to each of subroutines 10, 20, 30, and
thereby be able to further navigate and explore steps A comprising
steps A1, A2 each of which have options 1, 2, 3 . . . n 1b, 2b,
etc. . . . ) of the biotechnological process; selection by the user
of one or more parameters in each step (Step A, Step A1, Step B
Step B1 etc. . . . ) of the plurality of subroutines for the
biotechnological process to obtain a selected plurality of steps;
and storing (saving) the selected plurality of steps (and hence
selected subroutines) by the user in a memory, wherein the stored
selected plurality of subroutines comprise the at least one
user-generated workflow method file.
[0157] In some embodiments, the disclosure describes an in silico
method comprising a subsystem having a prescribed workflow of a
biotechnology process. A prescribed workflow typically comprises a
method file in a computer system comprising computer readable
instructions of a plurality of subroutines of a biotechnological
process, wherein performing each subroutine of the biotechnological
process would lead to a biotechnology product. For example, in a
non limiting example biotechnology process is a process for
cloning, a prescribed workflow of the disclosure may comprise
generating a method file in a computer system comprising computer
readable instructions for a plurality of subroutines such as, but
not limited to: amplifying fragments to use in cloning; inserting a
gene of interest into an entry clone; previewing entry clones;
using entry clones for cloning into a expression vector; create
expression clones; preview expression clones. A sequential
subroutine of such a prescribed workflow are presented to a user in
a GUI with each step as a link/button on the GUI which when
selected by a user leads a user to another GUI screen with further
details regarding that particular subroutine. For example, in the
process for cloning described above, selecting the subroutine of
"amplifying fragments to use in cloning" would lead a user to a
screen showing various steps of this subroutine. Each step may
further have sub-steps or several parameters each of which may be
displayed in a GUI using one or more buttons for each
sub-step/parameter, for example using certain primers for the
amplification which may be designed for insertion of the amplified
product into an entry clone plasmid.
[0158] In some embodiments, a prescribed workflow allows a user to
view in silico all steps of a workflow to make a biotechnology
product. A prescribed workflow also allows a user to in silico
navigate to each step and understand what each step entails. One or
more advantages of a prescribed workflow of the disclosure are
allowing users to view all steps required for a particular
biotechnology process thereby providing users with an overview of
the process requirements. This may especially help users that are
new and users that may not be familiar with that particular
biotechnology process. Other advantages of a prescribed workflow of
the disclosure is providing to a user a prescribed number of steps
to ensure that the workflow is standardized and results are assured
to have been processed in a similar fashion to provide consistency
between different executions of the method files/protocols.
[0159] In some embodiments, a prescribed workflow allows a user to
select or input parameters for each step and save and load the
workflow so a user may return to the workflow later to review it or
to provide it to other users so all users may perform the same
workflow.
[0160] Some example prescribed workflows of the disclosure include
a Prescribed Workflow for execution of Gateway.RTM. Cloning which
is shown for example in FIG. 4. Here, the users are notified via a
GUI of a "prescribed workflow" comprising a plurality of sequential
subroutines involved in performing Gateway.RTM. Cloning which
include for example: Subroutine 1. Amplify Fragments to use in BP
Reaction; Subroutine 2. Recombine Entry Clones by BP; Subroutine 3.
Preview Entry Clones; Subroutine 4. Add Entry Clones to use in LR
Reaction; Subroutine 5. Create Expression Clones by LR; and
Subroutine 6. Preview Expression Clones. As a user clicks (selects)
each step (in order or random order) conditions, parameters and
possible options for each step appear as additional GUI screens
with Navigation panels to choose, populate, save, return to
previous and/or enter one or more parameters or values.
[0161] Other non limiting examples of Prescribed Workflows of the
disclosure are TOPO.RTM. Cloning and ContigExpress.RTM. which are
depicted in FIG. 5 and FIG. 6 respectively. FIG. 5 depicts an
exemplary prescribed workflow of the disclosure for TOPO.RTM.
Cloning which comprises the following subroutines: Subroutine 1.
Amplify fragments to use in TOPO.RTM. reaction; Subroutine 2.
Create TOPO.RTM. clones; and Subroutine 3. Preview clones. Detailed
GUIs for each of these steps of the prescribed workflow are shown
in FIGS. 8A-8H.
[0162] FIG. 6 describes an example prescribed workflow of the
disclosure comprising a biological workflow for ContigExpress.RTM.
and comprises the following subroutines: Subroutine 1. View
Fragments; Subroutine 2. Trim ends; Subroutine 3. Trim vector
contaminations; Subroutine 4. Assemble contig; and Subroutine 5.
View contig. Detailed GUIs for each of these steps of the
prescribed workflow are shown in FIGS. 9A-9G.
[0163] GUIs of prescribed workflows for Gateway Cloning are shown
in FIGS. 7A-7O. GUI's of TOPO Cloning are shown in FIGS. 8A-8E and
GUI's for ContingExpress are shown in FIGS. 9A-9G. Details of these
figures (i.e., subroutines and steps of subroutines of these
workflows) are described in sections below.
[0164] Gateway.RTM. Cloning Technology is a rapid and highly
efficient method for the cloning and subcloning of DNA segments.
This system is based on the well-characterized bacteriophage
lambda-based site-specific recombination system (attLx
attR.quadrature.attBx attP). Gateway.RTM. Cloning is a 2-step
process. In the first step a sequence of interest containing attB
sites is recombined with a donor vector containing attP sites into
an entry clone, creating attL sites in the process. The second step
recombines the attL-containing entry clone with a destination
vector containing attR sites, generating an expression clone that
can be propagated and expressed in a range of host cells for a
given experiment. Additional detailed information on Gateway.RTM.
Cloning, see the Gateway.RTM. Technology User Guide or the
Gateway.RTM. Technology with Clonase.TM. II User Guide, available
for download from www.lifetechnologies.com/manuals.
[0165] A prescribed workflow of the disclosure for Gateway Cloning
may comprise 1) Step 1 comprising creating an entry clone which in
some embodiments may comprises creating an entry clone in the
Vector NTI Express Gateway.RTM. Cloning Tool by a) amplifying a
sequence or molecule of interest with attB containing primers
designed by the software; b) performing BP recombination with a
donor (pDONR) vector to generate an entry clone. This generates a
product described as the following:
PCR product(flanked by attB sites)+pDONR vector(with
attP).quadrature.Entry Clone(with attL)
[0166] In some embodiments, Step 1, comprising creating an entry
clone, may comprise the following alternative methods: [0167] i.
Selecting an existing attB-containing DNA molecule--From within the
Gateway.RTM. Cloning Tool, a user can select an already existing
attB-containing DNA molecule in the database, such as a
Gateway.RTM. Expression Clone or a pCMVSPORT6 library, for
recombination with a donor vector, to create an entry clone; or
[0168] ii. Constructing an entry clone by alternative molecule
construction methods--A user may alternatively construct his/her
own entry clone using other Vector NTI Express molecule
construction methods, ensuring that the created clone contains the
required attL1 and attL2 sites (labeled as features as described in
"Add Entry Clones to Use in LR Reaction"); and saving the molecule
in the database; and selecting it directly in the Gateway.RTM.
Cloning tool.
[0169] Step 2 of the prescribed workflow for Gateway Cloning
comprises creating an expression clone which may comprise using the
Gateway.RTM. Cloning Tool, entry clones created from any of the
above methods (described under step 1) are recombined with
destination (pDEST) vectors in an LR recombination reaction to
generate expression clones, which can drive expression of the
sequence of interest when transformed into host cells.
[0170] The Gateway.RTM. Cloning Tool contains settings and
functions for assembling a Gateway.RTM. construct using the
workflow described above, and for creating and managing
Gateway.RTM.. An example workflow of the disclosure comprising a
GUI would begin with Opening the Gateway.RTM. Cloning Tool.
[0171] There are several ways to open the tool and these may
comprise: 1) Clicking on the Gateway Cloning button on the main
toolbar; or 2) With a molecule open in Molecule Editor, right-click
in the Graphics or Sequence pane and select Launch Gateway; or 3)
Load an existing Gateway.RTM. Cloning project as described later in
describing the GUI for "Create, save, and load projects."
[0172] The tool window is composed of the following panes:
[0173] Gateway.RTM. Project Pane: This pane displays the name of
the current project, and includes controls for editing, saving, and
closing projects. It also lists any generated molecules for the
current project. Click on the Gateway Project button to display the
GUI pane as shown in FIG. 7A
[0174] The Task List pane: displays the list of tasks in the
selected project. Clicking on the Task button of the Gateway
Project Pane display the list as shown in FIG. 7B. As a user
navigates through the Gateway.RTM. Cloning workflow, the Task List
displays the current task, and allows a user to move between tasks
by clicking on a different task (see FIG. 7B).\ Current Task pane
as depicted in FIG. 7C displays the commands and settings for the
currently selected task in the project. As a user navigates through
the Task List workflow, the functions in this pane will change.
[0175] The Create, save, and load projects are tools for saving,
editing, and closing projects are located in the Gateway.RTM.
Project pane and are depicted in FIG. 7D. To save a new
Gateway.RTM. Cloning project, a user may click on Edit Project in
the Gateway.RTM. Project pane. In the dialog, a user may enter a
name and any description for the project. To save changes to a
project, a user may click on the Save Project button. To close a
project, a user may click on the Close Project button. If there are
unsaved changes, a user will be prompted to save the project before
closing.
[0176] To load an existing project, in Database Explorer, a user
may navigate to the Projects list as shown in FIG. 7E, double-click
on the Projects folder, select the Cloning Projects subfolder from
the Local Database, and double-click on the Gateway.RTM. Cloning
project in the list to open it.
[0177] The Task List displays the default Gateway.RTM. Cloning
workflow comprising the following steps as shown in the flowchart
in FIG. 8: Amplify Fragments to Use in BP Reaction; Recombine Entry
Clones by BP; Preview Entry Clones; Add Entry Clones to Use in LR
Reaction; Create Expression Clones by LR; Preview Expression
Clones.
[0178] Each task in the workflow is described here and illustrated
in FIGS. 7F-7O. For example, FIG. 7F depicts the Amplify fragments
to Use in a BP reaction GUI. The Amplify Fragments to Use in a BP
Reaction task is the first default task displayed when a user first
opens the Gateway.RTM. Cloning Tool. The first step in this task to
select the fragment(s) you want to amplify by PCR for use in a BP
cloning reaction. These fragments will be listed in the Fragments
to Amplify list.
[0179] Loading molecules or fragments in the Fragments to Amplify
list: With a molecule open in Molecule Editor, a user may
right-click in the Graphics or Sequence pane and select Launch
Gateway to load the entire sequence in the list. Alternatively,
with a molecule open in Molecule Editor, a user may select a
portion of the sequence in the Graphics or Sequence pane,
right-click, and select Launch Gateway to load only that part of
the sequence in the list. In another alternative embodiment, with
the Gateway.RTM. Cloning window open, a user can ensure the Amplify
Fragments to Use in BP Reaction task is selected and click on the
Add button under Fragments to Amplify to select a complete molecule
from the database. In some embodiments, to change the regions to
amplify in the selected molecules, a user may type a new range in
the From and To fields in the Fragments to Amplify subpane.
[0180] Amplification settings: With the fragment(s) loaded, a user
can select the desired amplification settings under PCR
Amplification Settings. The standard options are described in the
Table below.
TABLE-US-00001 Tm(C.) Enter limits in degrees Celsius for primer
melting temperature (Tm) (temperature at which 50% of primer is a
duplex) and the difference between Tm for sense and antisense
primers. % GC Enter limits in degrees Celsius for primer melting
temperature (Tm) (temperature at which 50% of primer is a duplex)
and the difference between Tm for sense and antisense primers.
Primer Length Defaults to 20-25, recommended for Gateway .RTM.
Primers DNA/RNA button Select the type of nucleotide sequence. Add
GGGG-attBx 5' The default attB extensions are for single Extensions
fragment cloning: attB1 for the sense primer and attB2 for the
antisense primer. Select from the dropdown list to replace the
defaults with other attB sequences for creating Entry Clones for
MultiSite Gateway .RTM. Cloning projects. Add generated Select this
checkbox to add the primers a user primers to oligo can generate to
the oligo list list
[0181] For additional amplification settings, a user may click on
Advanced. The advanced amplification settings are identical for all
PCR primers, and are similar to Primer Design described
previously.
[0182] After making the selections as described above a user may
click on the Amplify button. The next task pane will be displayed,
and the generated PCR product(s) will appear listed in the BP
Inserts subpane, and also listed in the Gateway.RTM. Project pane
as depicted in FIG. 7G.
[0183] FIG. 7H depicts the Recombine Entry Clones by BP. In the
Recombine Entry Clones by BP task pane, a user can modify the list
of fragments with attB sites and select a donor (pDONR) vector or
vectors with which to create entry clones.
[0184] BP Inserts: The fragments amplified by a user with attB
sites are listed in the BP Inserts list at the top of the pane. To
add a previously amplified fragment with attB sites, or a fragment
designed with attB sites by another means (e.g.,
restriction-ligation), a user may click on the Add button and
select the molecule from the database. To remove a molecule from
list, a user may select it and click on Remove. To clear the entire
list, a user may click on Clear All.
[0185] FIG. 7I depicts a pDONR vector which is a Gateway.RTM.
Cloning vector that contains attP sites, which are recombined with
the fragments containing attB sites to create entry clones. A
variety of pDONR vectors are sold by Life Technologies, and in
silico sequences for these are installed as part of the default
Vector NTI Express installation. To select a pDONR vector a user
may Click on the Add button and select the pDONR vector from the
database. To remove a vector from list, a user may select it and
click on Remove. To clear the entire list, a user may click on
Clear All.
[0186] Create the Entry Clone pane: When a user has chosen
selections, click on Create Entry Clone button will result in
opening of the Preview Entry Clones task pane an example of which
is depicted in FIG. 7J. Entry clones contain attL1 and attL2 sites,
and are used to generate expression clones via an LR reaction.
[0187] In the Entry Clones list: a User may Select a clone and
click on Save to Database to save it as a DNA molecule in the
database. A user may Double-click on a clone to display it in the
Preview window.
[0188] In the Preview window, magnifying tools depicted in FIG. 7K
may be used to zoom in and out of the molecule and a molecule may
be displayed as linear or circular by clicking on the buttons shown
in FIG. 7L.
[0189] Add Entry Clones to Use in LR Reaction task: After creating
entry clones, clicking on Add Entry Clones to Use in LR Reaction in
the Task List proceed to the next task in the workflow. Any entry
clone that is generated from the previous tasks in the workflow
will be listed in this window. To select new or additional entry
clones in the database, a user may click on the Add button and
select from the dialog box. To remove an entry clone from the list,
a user may select it and click on the Remove button.
[0190] Create Expression Clones by LR task: In the Create
Expression Clones by LR task pane, shown in FIG. 7M, a user can
modify the list of entry clones and select a destination (pDEST)
vector or vectors with which to create expression clones.
[0191] Entry Clones: Any entry clones that were generated from the
previous tasks in the workflow will be listed in the Entry Clones
list. To select new or additional entry clones in the database, a
user may click on the Add button and select from the dialog box. To
remove an entry clone from the list, a user may select it and click
on the Remove button. To clear the list, a user may click on Clear
All.
[0192] A pDEST vector such as the one depicted in FIG. 7N is a type
of Gateway.RTM. Cloning vector that contains attR sites, which are
recombined with the fragments containing attL sites to create
expression clones. A variety of pDEST vectors are sold by Life
Technologies, and in silico sequences for these are installed as
part of the default Vector NTI Express installation.
[0193] Preview Expression Clones: To select a pDEST vector: Click
on the Add button and select the pDONR vector from the database. To
remove a vector from list, select it and click on Remove. To clear
the entire list, click on Clear All.
[0194] For the step of Creating the Expression Clone after a user
makes selections as described above, a user may click on Create
Expression Clone. The Preview Expression Clones task pane will open
as depicted in FIG. 7O. A Preview Expression Clones task pane lists
all the expression clones created from the entry clones and the
destination vector(s) selected, and includes a preview window for
viewing an expression clone as shown in an example embodiment in
FIG. 7O.
[0195] In the Expression Clones list: a user may Select a clone and
click on Save to Database to save it as a DNA molecule in the
database or a user may Double-click on a clone to display it in the
Preview window.
[0196] Another set of GUIs are described and depicted in a
prescribed workflow relating to TOPO.RTM. Cloning in FIGS. 8A-8H.
Topo Cloning. TOPO.RTM. Technology is a fast, efficient way to
clone. The key to TOPO.RTM. Cloning is the enzyme DNA topoisomerase
I, whose biological role is to cleave and rejoin DNA during
replication. To harness this activity, vectors are linearized and
each end is conjugated with topoisomerase on the 3' phosphate. This
enables fast ligation of DNA sequences with compatible ends. After
5 minutes at room temperature, the enzyme is released, the ligation
is complete and the recombinant molecule is ready for
transformation into E. coli. Many Life Technologies expression
vectors are adapted for one-step TOPO.RTM. Cloning of PCR products
in both directional and non-directional formats. Other vectors
contain att recombination sequences exterior to the TOPO.RTM.
cloning sites so that cloned inserts are Ready for entry into the
TOPO.RTM. system.
[0197] TOPO.RTM. vectors can be grouped into three categories,
based on the nature of their ends: Zero-Blunt vectors have two
blunt ends and can accept blunt-ended DNA fragments, including
amplicons produced by a proofreading polymerase. Inserts are cloned
in both orientations.
[0198] T-A vectors have two ends with 3'-T overhangs. They can
accept products of PCR amplification with a Taq polymerase, whose
terminal transferase activity adds 3'A overhangs to the amplicon.
Inserts are cloned in both orientations.
[0199] In directional vectors one terminal is blunt ended and the
other has a 5'-GGTG overhang on the bottom strand. PCR products are
generated with a 5'-CACC extension on one end and this strand when
unwound is preferentially annealed to the vector overhang. More
than 90% of the clones are in the correct orientation and the time
spent in screening colonies is thereby reduced.
[0200] The presence of topoisomerase enzyme also helps protect
vector ends from degradation, particularly from contaminating
nucleases that may be present in ligase preparations. Moreover, the
avoidance of restriction site cutback for cloning PCR products
means that internal cleavage sites are not a problem.
[0201] Any linear, double-stranded DNA sequence of interest may be
cloned into a TOPO.RTM. vector using Vector NTI Express. In
addition, linear sequences with 3'-A overhangs, the products of PCR
amplification with a Taq DNA polymerase, may be cloned into
TOPO.RTM.-TA vectors. Such Taq-generated molecules can be generated
in silico using the TOPO.RTM. Cloning tool.
[0202] An exemplary prescribed workflow of the disclosure is
described by the TOPO.RTM. Cloning Tool. A user may begin this
workflow by Opening the TOPO.RTM. Cloning Tool. The TOPO.RTM.
Cloning Tool contains settings and functions for assembling a
TOPO.RTM. construct using the workflow described above, and for
creating and managing TOPO.RTM. Cloning projects. There are several
ways to open the tool: A user may Click on the TOPO Cloning button
on the main toolbar. Alternatively, with a molecule open in
Molecule Editor, a user may right-click in the Graphics or Sequence
pane and select Launch TOPO.RTM. Cloning. Still alternatively, a
user may Load an existing TOPO.RTM. Cloning project as described in
"Create, save, and load projects" buttons later. The TOPO cloning
tool window is composed of the following panes:
[0203] TOPO.RTM. Project Pane: The TOPO.RTM. Project pane as
depicted in FIG. 8A, displays the name of the current project, and
includes controls for editing, saving, and closing projects. It
also lists any generated molecules for the current project.
Clicking on the TOPO.RTM. Project button to display this pane.
[0204] The Task List pane as depicted in FIG. 8B displays the list
of tasks in the selected project. Clicking on the Task button to
displays the list shown in FIG. 8B. As a user navigates through the
TOPO.RTM. Cloning workflow, the Task List displays the current
task, and allows a user to move between tasks by clicking on a
different task.
[0205] Current Task pane is depicted in FIG. 8C and displays the
commands and settings for the currently selected task in the
project. As a user navigates through the Task List workflow, the
functions in this pane will change.
[0206] Create, save, and load projects are tools for saving,
editing, and closing projects are located in the TOPO.RTM. Project
pane which is depicted in FIG. 8 D. To save a new TOPO.RTM. Cloning
project, a user may click on Edit Project in the TOPO.RTM. Project
pane. In the dialog, a user may enter a name and any description
for the project. To save changes to a project, a user may click on
the Save Project button. To close a project, click on the Close
Project button. If there are unsaved changes, you will be prompted
to save the project before closing.
[0207] To load an existing project, in Database Explorer, a user
may navigate to the Projects list, as shown in FIG. 8 E and open
the Cloning Projects folder in the Local Database, and double-click
on the TOPO.RTM. Cloning project in the list to open it.
[0208] The TOPO Cloning workflow Task List displays the default
TOPO Cloning workflow and comprises as depicted in the prescribed
workflow shown in FIG. 6 the following steps: 1) Amplify Fragments
to Use in TOPO Reaction and 2) Create TOPO Clones.
[0209] This section describes each task in this prescribed
workflow:
[0210] Amplify Fragments to Use in the TOPO Reaction pane as
depicted in FIG. 8 F depicts the Amplify Fragments to use in TOPO
Reaction task which is the default task displayed when a user first
open the TOPO.RTM. Cloning Tool. The first step in this task to
select the fragment(s) a user want to amplify by PCR for use in a
TOPO cloning reaction. These fragments will be listed in the
Fragments list as shown in FIG. 8 F.
[0211] Load molecules or fragments in the Fragments list: With a
molecule open in Molecule Editor, right-click in the Graphics or
Sequence pane a user may select Launch TOPO Cloning to load the
entire sequence in the list. Alternatively a user may, with a
molecule open in Molecule Editor, select a portion of the sequence
in the Graphics or Sequence pane, right-click, and select Launch
TOPO Cloning to load only that part of the sequence in the list.
Yet alternatively, with the TOPO.RTM. Cloning window open, a user
may ensure the Amplify Fragments to Use in TOPO Reaction task is
selected and click on the Add button under Fragments to select a
complete molecule from the database.
[0212] To change the regions to amplify in the selected molecules,
a user may type a new range in the From and To fields in the
Fragments to Amplify subpane. PCR Amplification Settings with the
fragment(s) loaded, select the desired amplification settings under
PCR Amplification Settings. The standard options are described in
the Table below.
TABLE-US-00002 Tm(C.) Enter limits in degrees Celsius for primer
melting temperature (Tm) (temperature at which 50% of primer is a
duplex) and the difference between Tm for sense and antisense
primers. % GC Enter limits in degrees Celsius for primer melting
temperature (Tm) (temperature at which 50% of primer is a duplex)
and the difference between Tm for sense and antisense primers.
Primer Length Defaults to 20-25, recommended for TOPO .RTM. Primers
DNA/RNA Select the type of nucleotide sequence. button Add
generated Select this checkbox to add the primers a user can
primers to generate to the oligo list oligo list
[0213] For additional amplification settings, a user may click on
Advanced. The advanced amplification settings are identical for all
PCR primers, and are similar to Primer Design described
previously.
[0214] After making the selections as described above a user may
click on the Amplify button. The next task pane will be displayed,
and the generated PCR product(s) will appear listed in the Inserts
subpane, and also listed in the TOPO.RTM. Project pane as depicted
in FIG. 8G.
[0215] Create TOPO.RTM. Clones: In the Create TOPO.RTM. Clones task
pane as shown in FIG. 8H, a user can modify the list of fragments
and select a vector or vectors with which to create TOPO.RTM.
clones
[0216] Inserts: The fragments amplified are listed in the Inserts
list at the top of the pane. To add a previously amplified fragment
or a fragment designed with the necessary overhangs by another
means, a user may click on the Add button and select the molecule
from the database. To remove a molecule from list, a user may
select it and click on Remove. To clear the entire list, a user may
click on Clear All.
[0217] Vectors: To select a vector: A user may click on the Add
button and select the vector from the database. To remove a vector
from list, a user may select it and click on Remove
[0218] Create the TOPO.RTM. Clone: when all selections have been
made a user may click on Create TOPO.RTM. Clone. The Preview Clones
task pane will open (not expressly depicted here).
[0219] FIGS. 9A-9G depicts a prescribed workflow GUI describing the
exemplary workflow shown in FIG. 10 relating to
ContigExpress.RTM..=Accordingly a default display of all steps will
appear upon launching ContigExpress.RTM.. Step 1 comprises Lauching
Contig Express.RTM. which brings up the screen as shown in FIG. 9A
which shows all the default steps (Subroutines) of ContigExpress
prescribed workflow including 1. View Fragments 2. Trim ends 3.
Trim vector contaminations 4. Assemble contig and 5. View contig as
shown in the left navigation pane in FIG. 9A.
[0220] GUI of Step 2 is shown in FIG. 9B which shows Selecting ABI
files from the file system
[0221] Selecting the first subroutine of the prescribed workflow of
View Fragments brings the GUI screen as shown in FIG. 9C. Selecting
the second subroutine of the prescribed workflow of Trim Ends of
all Fragments brings the GUI screen as shown in FIG. 9D. Selecting
the third subroutine of the prescribed workflow of Trim Vector
Contaminants brings the GUI screen as shown in FIG. 9E. Selecting
the fourth subroutine of the prescribed workflow of Assemble Contig
brings the GUI screen as shown in FIG. 9F. Selecting the fifth
subroutine of the prescribed workflow of View Contig brings the GUI
screen as shown in FIG. 9G.
[0222] In some embodiments, the disclosure describes an in silico
method comprising a subsystem for the creation of a customized
workflow of a biotechnology process. In some embodiments, creation
of an in silico customized workflow may comprise: generation by a
user of at least one method file in a computer system, the method
file comprising, computer readable instructions of a plurality of
steps of a biotechnological process, wherein the user inputs and/or
selects a plurality of parameters associated with each step of the
biotechnological process; and performing the biotechnological
process comprising executing the at least one method file
comprising computer readable instructions by the computer system to
obtain at least one biotechnology product. In some embodiments of
this method, the step of generation by a user of a method file
comprising a plurality of steps may comprise generation of steps
that may be in a sequential order or may alternatively comprise
generation of steps that are not in any particular order.
Typically, all the parameters associated with each step (ordered or
random steps) of a method file are selected or inputted by a user
prior to execution of the method file. In some embodiments, a
navigation panel facilitates a user to input/select parameters
associated with each step. In some embodiments, selection or input
of user defined parameters in steps may be based on available tools
and subsystems in a computer system. In some embodiments, one or
more steps may involve inclusion of data which may be user input
data. In some embodiments a step may involve pre-processing of data
by the system.
[0223] In some examples, a user may use a basic prescribed workflow
and customize it with user input of various parameters and/or
values, save such a workflow as a customized prescribed workflow of
the disclosure. A user may save a workflow (prescribed or
customized) locally such as on a local hard-drive, a floppy disk, a
USB stick, in a .txt file, in an .html format, and/or even be able
to send the saved protocol via email.
[0224] Prescribed and customized workflows of the disclosure also
allow user to analyze an in silico method and steps thereof,
wherein the analyzing is executed by the computer system. In some
embodiments an in silico method of the disclosure may further
comprise generating reports of the workflow wherein the generating
reports is executed by the computer system. In some embodiments,
reports may be generated based on analysis of the steps customized
by a user.
[0225] In some embodiments, the disclosure describes in silico
methods comprising a subsystem for tracking the progress of a
biotechnology process. In some embodiments tracking progress of a
biotechnology process may comprise user navigation during the
execution of a workflow.
[0226] In some embodiments, the present disclosure describes in
silico methods for a biotechnology process for achieving an optimal
end result. Accordingly a user may change/customize one or more
steps parameters using a GUI to input user selections or data and
arrive at an optimized procedure for a biotechnology process. In
some embodiments, a user may check the in silico method by
performing experiments in the lab. The ability to save each
parameter change as a separate in silico method enables a user to
review results of each method and arrive at an optimum method.
[0227] Some non limiting examples of applications of the in silico
methods of the disclosure may be to provide other non-skilled users
an optimized in silico procedure for a biotechnology process; to
provide one standardized customized method to different users to
perform similar biotechnology processes to provide a consistent
workflow across diverse users; to provide a method whereby one
parameter at a time may be varied to arrive at an efficient
method.
[0228] In some embodiments, an in silico method of the disclosure
may comprise combining a plurality of workflows (prescribed or
customized) to arrive at custom production workflow. Accordingly,
several individual workflow protocols may be combined to
consistently produce a biotechnology product, wherein the end
product of one workflow (of the plurality of workflows) may for
example be used as a start product for another workflow. In a
non-limiting example an in silico workflow for making a modified
host system in silico may comprise combining at least one workflow
for cloning; at least one workflow for making a vector; at least
one workflow for selection to arrive at a modified host system
expressing a gene of interest.
[0229] It will be understood by one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are readily apparent
from the description of the invention contained herein in view of
information known to the ordinarily skilled artisan, and may be
made without departing from the scope of the invention or any
embodiment thereof.
Example 1
[0230] The following example illustrates one specific aspect of the
methods and computer systems of the invention.
Overview of VectorDesigner
[0231] VectorDesigner is a secure, online tool for clone
construction and management. Using VectorDesigner, you can import,
view, construct, analyze, and save DNA and protein sequences in a
Web-based environment, and then export your molecule constructions
as standalone files to share with colleagues.
[0232] VectorDesigner provides a secure Web-based database for
storage and management of your clone sequences and designs. It
includes interactive tools for identifying ORFs and restriction
sites, translating sequences, generating PCR primer designs,
searching public sequence databases, and performing other types of
molecule analysis. You can design complex cloning experiments using
proprietary Gateway.RTM. and TOPO.RTM. technologies or common
methodologies such as restriction-ligation and PCR. You can analyze
your sequences using other Invitrogen tools (BLOCK-iT.TM. RNAi
Designer, CloneRanger.TM., OligoPerfect.TM. Designer, LUX.TM.
Designer) and import the results back into VectorDesigner.
[0233] VectorDesigner is based on VectorNTI Advance.TM., a software
suite for sequence analysis and molecular data management,
available for Windows.RTM. and Macintosh.RTM. operating systems.
Files created and saved using VectorDesigner can be opened
seamlessly in VectorNTI Advance.TM., which provides more powerful
analysis tools and enhanced databasing capabilities. See FIG. 5 for
a comparison of the functionalities of VectorDesigner.TM. and
Vector NTI Advance.TM.. See also, Invitrogen's Bioinformatics
Software Web page for more information about VectorNTI
Advance.TM..
Database Operations VectorDesigner Database Browser
[0234] The VectorDesigner database contains DNA, RNA, and protein
molecule files in a hierarchy of folders and subfolders. The
Database Browser provides access to the entire contents of the
database.
[0235] Click on the Browse Database tab in VectorDesigner to view
the Database Browser. The Browser window is divided into two main
panes: the "Folder tree" and the "Molecules list".
[0236] The Folder tree displays the database folder structure. Use
the folder tree to navigate through the database. Click on a folder
name to view its contents. Click on the .+-.button next to each
folder name to expand or collapse the folder. Note: The Folder tree
is only visible if you approved the signed security certificate
when VectorDesigner first opens.
[0237] The Molecules list displays the contents of the currently
selected folder. Click on a molecule name in the list to open the
molecule file. Select the checkbox next to the molecule name to
rename, move, or delete the molecule file. Click on the Import
button to enter a new molecule sequence or import a molecule file
from another source.
Database Folders
[0238] The Database Browser window displays the folder structure of
the VectorDesigner database. The database has five main
folders--three user folders (DNA/RNAs, Proteins, and Primers) and
two read-only folders (Invitrogen Vectors and Examples)
User Folders
[0239] User folders are private, secure folders that contain
molecule files that you create or modify (e.g., DNA/RNAs; Proteins;
Primers). The contents of these folders are created and controlled
by the user, are keyed to your user name and password, and cannot
be viewed by other users. The molecule files in these folders can
be edited, renamed, deleted, moved, and exported for collaboration
with other users.
[0240] You can create new folders within the three main user
folders. However, you cannot delete or move the three main user
folders, and you cannot add new main user folders.
[0241] Each main user folder can contain only molecules of the
specified type (DNA/RNAs, Proteins, and Primers). For example, you
cannot store DNA molecule files in the Proteins folder.
Read-Only Folders
[0242] Read-only folders contain molecule files created by
Invitrogen: Invitrogen Vectors, which contains sequences and maps
of vectors sold by Invitrogen, including Gateway.RTM. and TOPO.RTM.
vectors; and Examples, which contains sequence-verified example
files of common DNA, protein, and primer molecules. These folders
and files cannot be modified or deleted and are accessible to all
users. You can copy the molecule files in these folders into your
private user folders and edit them.
Editing Folders
[0243] You can add folders within the main user folders. Click on a
folder name to select it and click on the Create a New Folder
button to create a subfolder within that folder. To delete a folder
that you created, click on the folder name to select it and click
on the Delete Folder button. Note: Deleting a folder will also
delete all its contents.
[0244] You can rename the main user folders or any of their
subfolders. Click on the folder to select it, and click on the
Rename Folder button. Enter the new name in the pop-up box and
click on OK. Note that renaming a user folder will not change the
file type restriction for that folder.
Folder Restrictions
[0245] User folders have the following restrictions: The DNA/RNAs
folder and Proteins folder can contain up to 100 molecules each;
The Primers folder can contain up to 1,000 molecules. DNA and
protein molecules are restricted to 350,000 base pairs or amino
acids in length; Primers are restricted to 250 bases in length;
Each folder can contain only molecules of the specified type (e.g.,
you cannot store DNA molecule files in the Proteins folder); Each
main user folder can have only 10 subfolders; Molecules with the
same name cannot be saved in the same folder.
Database Capacity
[0246] The VectorDesigner database has the following limits: The
DNA/RNAs folder and Proteins folder can contain up to 100 molecules
each; The Primers folder can contain up to 1,000 molecules; DNA and
protein molecules are restricted to 350,000 base pairs or amino
acids in length; Primers may be restricted in size (e.g., 250 bases
in length).
Molecule Files
[0247] A molecule file contains all the information about a
molecule, including sequence, name, description, features,
references, comments, analysis, etc. Molecule files for DNA/RNA and
protein molecules are based on the GenBank/GenPept format, which is
an ASCII text-based format, and can be exported as stand-alone
files in a variety of formats.
[0248] Molecule files that are created, imported, or modified by
you are stored in private user folders in the database and are
accessible using your user name and password. Example molecule
files created by Invitrogen are stored in read-only folders
(Invitrogen Vectors and Examples) and are viewable by everyone.
[0249] The Database Browser window provides access to all the
molecule files in the database. Using the Browser, you can open,
rename, move, delete, import, and export the molecule files in your
user folders, and you can export molecule files in the read-only
folders. Molecule File Types
[0250] Molecule files in VectorDesigner can contain DNA or RNA
sequences, protein sequences, or primer sequences. The type of
molecule determines the information contained in the molecule file,
which user folder it is stored in, and which viewer it is displayed
in. DNA and RNA Molecule Files contain circular or linear
nucleotide sequences, are stored in the DNA/RNAs folder in the
database, and are displayed in the Molecule Viewer. Protein
Molecule Files contain amino acid sequences, are stored in the
Proteins folder in the database, and are displayed in the Molecule
Viewer. Primer Files contain DNA or RNA primer sequences, are
stored in the Primers folder in the database, and are displayed in
the Edit Primer Properties window.
[0251] Molecule Files in the Database Browser: Molecule files are
listed in the right pane of the Database Browser window. Click on a
folder name in the Browser to display the molecule files in that
folder. If you make changes to the molecules list in the Browser
and those changes are not updated in the Browser window, click on
the Reload button to refresh the list. If you have more than 50
molecules in the list, use the scroll buttons to scroll through the
list.
Opening Molecules
[0252] To open a molecule file: 1. Navigate to the appropriate
folder in the Database Browser; and 2. Click on the molecule name
in the molecules list. For DNA/RNA and protein molecules, the
Molecule Viewer window for that molecule will open. For primers,
the Edit Primer Properties window will open.
Saving Molecules
[0253] You can save changes that you make to molecule files.
Changes to molecules opened from read-only folders must be saved
under a different file name in your private user folders. Unsaved
DNA/RNA/protein molecules are flagged with a "*" in the Molecule
Viewer title bar.
Saving DNA/RNA and Protein Molecules
[0254] To save a DNA/RNA or protein molecule in the Molecule
Viewer, go the Molecule menu and select Save or click on the Save
button on the main toolbar. To save a molecule with a different
file name or in a different location, go the Molecule menu and
select Save As or click on the Save As button on the main toolbar.
The Save As dialog will open. In the dialog, rename the file and/or
select or create a new folder to save it in.
[0255] Saving Primer Molecules To save a standalone primer file, in
the Edit Primer Properties dialog, select the Rename or the
Overwrite option button, and click on Save. If you selected Rename,
the primer file will be automatically saved with a numerical
extension appended to the file name (e.g., a file named Primer will
saved as Primer (1)).
Importing Molecules
[0256] You can import molecule and primer sequences and other
information into VectorDesigner in a variety of file formats,
including: GenBank/GenPept; EMBL; SWISS-PROT; Vector NTI.RTM. (uses
the GenBank format); FASTA; Plain text.
Exporting Molecules
[0257] You can export molecules from VectorDesigner in a variety of
file formats. You can export the data for one or more molecules at
a time from the Database Browser, or you can export the data for
the current molecule loaded in the Molecule Viewer. You can export
one or more molecules from the Database Browser to a file (text
format) or to a browser window (HTML format). You can export the
data for a DNA, RNA, or protein molecule displayed in the Molecule
Viewer to a variety of formats.
Export to Vector NTIO
[0258] You can export the molecule data from VectorDesigner to
Vector NTI.RTM.. In the Molecule
[0259] Viewer window, click on the Export to Vector NTI button on
the main toolbar or select the command from the Molecule menu.
[0260] You will be prompted to save the file (.gb extension for
DNA/RNA files, .gp extension for protein files) or automatically
launch Vector NTI.RTM. and display the molecule in the application
window. Note that Vector NTI.RTM. software must be installed on
your computer to automatically launch the application.
Export to GIF
[0261] You can export the molecule image as it is displayed in the
Molecule Viewer as a GIF image. Note: This command will export only
the current view of the molecule. If the displayed information
(sequence, graphics, text, etc.) is cut off at the margins of the
panes in the Molecule Viewer, the data will appear cut off in the
resulting image. Be sure to configure your Molecule Viewer panes as
desired for the resulting image.
Export Format
[0262] The exported information will vary depending on the export
format you select. Each database format (GenBank, EMBL, Vector
NTI.RTM., etc.) will include formatting and information compatible
with that database. All formats include the molecule sequence. The
available formats are: DNA/RNA molecules--GenBank, EMBL, and FASTA;
Proteins--GenPept, Protein FASTA, SWISS-PROT; Primers--GenBank,
EMBL, FASTA, and Tab-delimited
Moving Molecules
[0263] To move one or more molecule files the user can (1) Navigate
to the appropriate user folder in the Database Browser; (2) Select
the checkbox(es) next to the molecule name(s); (2) Click on the
Move button; and (3) In the Pick a Folder window, navigate to the
desired folder and click on OK. You can only move molecule files
within the same main user folder. For example, you cannot move DNA
molecule files into the Proteins folder. Molecules with the same
name must be stored in separate folders.
Deleting Molecules
[0264] You can delete molecule files from the user folders of the
VectorDesigner database. Note that deleted molecule files cannot be
recovered from the database, and you will be prompted to confirm
the deletion. To delete one or more molecule files the user can (1)
Navigate to the appropriate user folder in the Database Browser;
(2) Select the checkbox(es) next to the molecule name(s); (3) Click
on the Delete button; (4) Click on OK to confirm the deletion.
Creating New Molecules
[0265] You can create a new molecule based on the molecule
currently displayed in the Molecule Viewer. You can create a new
molecule from a selected area of the existing molecule, such as a
restriction fragment, or from the whole molecule.
[0266] For DNA or RNA molecules, you can create DNA/RNA molecules
that are the reverse complement of the existing molecule or you can
create protein molecules from a translation of the sequence. In the
Molecule Viewer, click on the Create New Molecule button on the
main toolbar or select the command from the Molecule menu. In the
dialog, enter a name for the new molecule in the Name field, and a
description (if any) in the Description field. Next, specify which
part of the existing molecule to use as the basis for the new
molecule. If you selected or marked a region of the existing
molecule before you opened the dialog, the Selection or Mark
options will be available and selected. Otherwise, select Molecule
to select the whole molecule or Specified Range to enter the
sequence range in the From and To fields. DNA/RNA molecules only:
Select the Reverse Complement checkbox to create a molecule from
the complementary sequence. Select Translate to create a protein
molecule from a translation of the sequence. When you have made
your selections, click on OK. The new molecule will be created and
displayed in a new Molecule Viewer window.
Renaming Molecules
[0267] You can rename the molecule files in the user folders of the
VectorDesigner database. To rename a molecule file: Navigate to the
appropriate user folder in the Database Browser; Select the
checkbox next to the molecule name in the molecules list; Click on
the Rename button; Enter the new file name in the pop-up window and
click on OK.
Revert Changes
[0268] You can undo all changes that you have made to a DNA/RNA
molecule since it was last saved. Go to the Molecule menu and
select Revert Changes to execute this command.
Molecule Viewer
[0269] The Molecule Viewer displays all the information in the
database for DNA/RNA molecules and protein molecules. (Information
for primers is displayed in the Edit Primer Properties dialog.)
When you open a DNA/RNA or protein molecule file from the Database
Browser, the Molecule Viewer will launch and display the
molecule.
[0270] The Molecule Viewer can display: The molecule sequence; A
graphical representation of the molecule; Information about the
molecule, including a list of molecule features; The results of
analysis performed on the molecule.
[0271] The Viewer can be divided into three main panes, the Text
Pane, the Graphics Pane, and the Sequence Pane, each with its own
set of tools and resources. The Text Pane provides database
information about the molecule, including a molecule description,
and list of molecule features, database keywords for the molecule,
references to literature/other materials, links to resources
related to the molecule, fields for user comments, and information
about any analysis performed on the molecule (restriction sites,
primer designs, etc.). The Graphics Pane displays a feature map of
the molecule, and includes interactive tools for adding and editing
features, highlighting and marking areas of the sequence, and
displaying the molecule. An Analysis Pane can also be displayed in
the Graphics Pane.
[0272] The Sequence Pane displays the entire nucleotide/amino acid
sequence, and includes interactive tools for editing and marking
the sequence, adding features, rearranging sequence elements, and
copying and pasting the sequence. In this pane you can also toggle
between a view of the sequence and a detailed view of the molecule
feature list. A Feature List can also be displayed in the Sequence
Pane.
[0273] The Text Pane in the Molecule Viewer contains textual
information about the molecule, including a general description,
comments, references, descriptions of molecule features, and the
results of any analysis. To change the molecule description,
comments, associated genes, keywords, references, etc. in the Text
Pane, use the Molecule Properties dialog.
[0274] The Text Pane is structured as a directory tree. Click on
the .+-.buttons to expand or collapse the branches of the tree.
Alternatively, right-click on the branch and select Expand Branch
or Collapse Branch.
[0275] Copying Text: To copy and paste text from the Text Pane:
Click on the branch or feature that you want to copy. To select
multiple branches/features, use Shift-Click or Control-Shift key
commands. To select all branches and features, right-click anywhere
in the Text Pane and select Select AH. Next, right-click on the
selection and select Copy Text. The text will be copied to the
computer clipboard. Paste the text into the application of your
choice.
Link Mode
[0276] You can link the display in the Graphics and Sequence Panes
to the folders that are open in the Text Pane control using the
Link Mode command. When linked, information from the open folders
in the Text Pane is displayed in the Graphics and Sequence Panes,
while information in closed folders is not displayed. (Note that
the molecule name and length is always displayed in the Graphics
and Sequence Panes.)
Protein Parameters (Protein Molecules Only)
[0277] The Text Pane for protein molecules includes a table of
Protein Parameters, which lists some of the biochemical properties
of the protein, such as molecular weight, A280 absorbance,
isoelectric point, etc. These properties are automatically
calculated by VectorDesigner from the amino acid sequence.
Graphics Pane
[0278] The Graphics Pane in the Molecule Viewer contains a
graphical representation of the DNA, RNA, or protein molecule,
highlighting the results of any analyses such as ORFs, restriction
sites, and other defined features. It includes a toolbar below the
pane. Additional tools are located on the View menu and on the
context menu if you right-click in the Graphics Pane. Defined
features in the molecule are shown as colored bars in the Graphics
Pane. Directional features (such as coding DNA sequences, or CDSs)
are shown as bars with directional arrows. Open reading frames are
shown as thin directional arrows. Restriction endonuclease sites
are labeled with the name of the enzyme.
Circular and Linear Display of DNA/RNA Molecules
[0279] For circular DNA/RNA molecules (as defined in the Molecule
Properties dialog), you can toggle between a circular and linear
display. Click on the Display as Circular button or the Display as
Linear button below the Graphics Pane, or select the commands from
the View Graphics Map submenu.
[0280] Note that this only changes the molecule display. To change
the actual molecule structure from circular to linear or vice
versa, use the Molecule Properties dialog.
Showing Labels
[0281] To show and hide labels in the Graphics Pane, click on the
Show/Hide Labels button below the Graphics Pane, or select the
command from the View>Graphics Map submenu. For molecules with
more than 80 features, labels are hidden by default.
Link Mode
[0282] You can link the display of features (including ORFS,
restriction sites, etc.) in the Graphics Pane to folders that are
open in the Text Pane using the Link Mode command. When linked,
features of open folders in the Text Pane are displayed in the
Graphics Pane, while features in closed folders are not
displayed.
Standard Arrangement
[0283] If you change the displayed labels and features in the
Graphics Pane (e.g., using Link Mode), you can reconfigure the pane
to make best use of the available space. Go to the View Graphics
Map submenu and select Standard Arrangement.
Sequence Pane
[0284] The Sequence Pane in the Molecule Viewer shows the sequence
of a DNA/RNA or protein molecule in a scrollable, wrap-around
field, with the starting base/amino acid number of each line shown
to the left. The Sequence Pane uses standard code letters to
indicate the bases/amino acids in the sequence. For DNA molecules,
by default, both the direct and complementary strands are shown.
(See Changing the Sequence Display, below.) Hold your cursor over
the sequence to display a popup box showing the base/amino acid
number at that point in the sequence.
[0285] The Feature List is also displayed in the Sequence Pane.
Click on the Feature List button to the left of the Sequence Pane
to view the Feature List. Click on the Sequence Pane button to
return to a view of the sequence.
[0286] To change how the sequence is displayed in the pane,
right-click in the Sequence Pane and select Sequence Properties.
Various sequence and feature representation styles are available.
In the Sequence Properties dialog, you can select the following
display options:
Types Filter
[0287] To filter the types of features highlighted in the Sequence
Pane, right-click in the pane and select Types Filter. In the
dialog, all available features will be selected. Deselect the
checkboxes next to the filters that you do not want to view in the
Sequence Pane, and click on OK to make the changes.
Link Mode
[0288] You can link the display of features (including ORFs,
restriction sites, etc.) in the Sequence Pane to the folders that
are open in the Text Pane using the Link Mode command. When linked,
features of open folders in the Text Pane are displayed in the
Sequence Pane, while features in closed folders are not displayed.
To enable this feature, click on the Link Mode button on the main
toolbar or select the command from the View menu.
Analysis Pane
[0289] The Analysis Pane displays graphical plots of a variety of a
DNA and protein sequence analyses. You can display multiple plots
at a time in the Analysis Pane. The available analyses depend on
the molecule type (DNA/RNA or protein).
[0290] The Analysis Pane and the Graphics Pane are displayed in the
same pane in the Molecule Viewer. The Graphics Pane is displayed by
default. To display the Analysis Pane, click on the Analysis Pane
button below the Graphics Pane. To return to a view of the Graphics
Pane, click on the Graphics-Pane button.
Graph Format
[0291] The graphs in the Analysis Pane display different
physiochemical properties of the sequence. Many of properties are
based on parameters like charge that exert effects over distance.
Other properties represented in the plot depend on the way adjacent
bases/amino acids fold in 3-dimensional space, which is a function
of the sequence itself.
[0292] The vertical (Y) axis in the graph shows the values of the
analysis results; the horizontal (X) axis displays either numerical
positions in the sequence or residues. At any point along the
sequence, the Y value is derived not just from the specific residue
at that point but also from adjacent residues. Each analysis
algorithm uses an optimum window of adjacent residues to calculate
the value for a point. You can adjust this window size in the Plot
Properties dialog (see below).
[0293] Note: No values may be calculated at the beginning and end
of the sequence if there are not enough bases/amino acids to the
left or right of each base/amino acid for the algorithm to
calculate a value. To calculate values for those regions, you can
reduce the window size in the Plot Properties dialog.
Plots Setup
[0294] Use the Plots Setup dialog to select and arrange the
analysis graphs to display in the pane. To open the dialog, click
on the Plots Setup button below the Analysis Pane or select the
command from the right-click menu.
[0295] In the Plots Setup dialog, the available analyses are listed
in the top window and the selected graphs are listed in the bottom
window. Analysis graphs are displayed in panels. You can add one or
more analyses to a panel, and display multiple panels in the
Analysis Pane.
Plot Properties
[0296] The Plot Properties dialog controls how each plot is
displayed in the graph. To open the dialog, right-click on an graph
in the Analysis Pane and select Plot Properties. The dialog is
divided into three tabs. When you have made your selections, click
on OK.
Diagram Tab
[0297] Click on the Graph Color button to open a dialog in which
you can select a plot color and/or adjust the Red-Green-Blue (RGB)
values of the color. Select the Draw Type from the dropdown list.
Min-Max-Average displays the calculated minimum, maximum, and
average values over each analysis region within the sequence as
levels of shading along the line of the graph.
[0298] Under Preprocess Type, select Linear Interpolation to
provide a linear interpolation of the graph line, or No
Preprocessing to display the line without interpolation.
Params Tab
[0299] Window Size is the size of the processing "window" used to
scan the sequence for analysis. Enter a number of bases/amino acids
in the Window Size field (see example below). Step Size is the
number of bases/amino acids in a sequence that constitute an
analysis point in the plot. Enter number of bases/amino acids in
the Step Size field (see example below). Example: If you select a %
GC Content analysis with a window size of 21 and a step size of 1,
the GC content percentage will be calculated for a 21-base region
centered on each base in the sequence (10 bases on either side of
the base). A step size of 5 would calculate the percentage for a
21-base region centered on each 5-base region in the sequence.
Info Tab
[0300] This tab provides information on the type of analysis in the
plot, including any references to external literature.
Feature List
[0301] The Feature List is list of the defined features in the
molecule in an easy-to-read table format.
[0302] The Feature List is displayed in the Sequence Pane. Click on
the Feature List button (Efs) to the left of the Sequence Pane to
view the Feature List. Click on the Sequence Pane button (IiIcJI)
to return to a view of the sequence. Click on a column header in
the Feature List table to sort the list by that column. Right-click
on a feature in the list and select Edit Feature Properties to open
the Add/Edit Feature dialog. Right-click on a feature in the list
and select Copy Text to copy the feature information to the
computer clipboard in a tab-delimited format. Right-click on a
feature in the list and select Open Link to access a variety of
links to online databases with information about the feature. Note
that links are available for only certain types of imported
molecules.
Window Manager
[0303] Use the Window Manager dialog to switch between multiple
open Molecule Viewer windows. To open the Windows Manager, go the
Windows menu and select Windows.
[0304] All open Molecule Viewer windows will be listed in the
dialog. To bring a window to the front, double-click on it in the
list. To close a window, select it in the list and click on Close
Windows. To close multiple windows, select them using Control+Click
and Shift+Click key commands and click on Close Windows. Click on
Exit to close the manager.
Molecule Features
[0305] Using VectorDesigner, you can label the various features in
a DNA/RNA or protein molecule, including promoter regions, open
reading frames, binding sites, epitopes, or any other region of
interest. The Feature Map folder in the Text Pane of the Molecule
Viewer contains a list of labeled features. The Imported Features
Not Shown on Map folder contains a list of unlabeled features. You
can label as many features in a molecule as you want. Features are
listed in the Text Pane and shown in the Graphics and Sequence
Panes.
Adding Features
[0306] Note: You can label an open reading frame, restriction
fragment, or primer as a feature. See Annotating Analysis.
[0307] To add a feature to the Feature Map folder: Select the part
of the sequence that you want to label as a feature, or mark
multiple areas of the sequence that you want to label as a single
feature. Click on the Add Feature button or select the command from
the Edit menu. (You can also right-click in the Graphics or
Sequence Pane and select Add Feature from the context menu.) In the
Add/Edit Feature dialog, the Feature Type field lists the available
feature types in the database for the molecule. Select a feature
type from the list. If you cannot find the precise type you are
looking, select Misc. Feature. Note that you cannot add new feature
types in VectorDesigner. Enter a name for the feature in the
Feature Name field. Select the format to use for defining the
sequence region: Use Start-End Format or Use Start-Length Format.
If you selected the feature region or marked multiple regions in
the sequence before opening the dialog, the start and
length/endpoint(s) of the feature will be automatically entered in
the dialog. To change the region, enter the start and
length/endpoint(s) in the fields. For features with multiple
components (i.e., internal start and endpoints), select
Multi-component and enter each start and length/endpoint in the
field. Use the following format: <start1> . . .
<length/endpoint1>, <start2 . . . length/endpoint2>,
etc. Click on Reset to Selection to undo any changes you may have
made to a preselected sequence region. Click on Reset to Mark to
undo any changes you may have made to a marked sequence region.
Select the Complementary checkbox if the feature is located on the
complementary molecule strand. Note: VectorDesigner uses the
currently accepted convention for calculating the coordinates of
complementary features. All coordinates are given as if on the
direct strand, from left to right in the sequence. Enter a
description for the feature in the Description field. When you have
made your selections, click OK to add the feature.
[0308] When you click on OK, information about the feature will be
added to the Feature Map in the Text Pane, and the feature will be
flagged in the Graphics and Sequence Panes as described below.
Viewing and Selecting Features
[0309] Text Pane: In the Text Pane, labeled features are listed by
type under the Feature Map folder. Note that many of the feature
types in VectorDesigner are mapped to keys in the GenBank and
GenPept databases.
[0310] The user may click on the +button next to each feature type
to view all the features of that type. Click on the +button next to
each feature name to view the information for that feature,
including sequence location, length, description, and any Web links
Features with multiple components will list each component
separately under the feature information. Double-click on the
feature name in the Text Pane to display the feature in the
Graphics and Sequence Panes.
[0311] Graphics Pane: Features are displayed in the Graphics Pane
by large colored arrows. Hold your cursor over feature arrow to
display a popup information box for that feature. Click on a
feature arrow to select that feature in the Sequence Pane, or
right-click on the arrow and select Find in Tree to locate the
feature in the Text Pane.
[0312] Sequence Pane: In the Sequence Pane, features are marked by
colored bars above the sequence. Click on feature bar in the
Sequence Pane to select the feature in the Graphics Pane, or
right-click on the arrow and select Find in Tree to locate the
feature in the Text Pane.
[0313] Feature List: The Feature List, displayed in the Sequence
Pane, lists each feature in the molecule in an easy-to-read table
format. Double-click on the feature name in the Feature List to
display the feature in the Graphics Pane.
Editing Features
[0314] To edit a feature: Right-click on the feature in the Feature
Map folder, Feature List, or Imported Features Not Shown in Map
folder; and Select Edit Feature Properties from the context menu.
This opens the Add/Edit Feature dialog.
Deleting Features
[0315] You can delete the feature definition and information
without removing the actual sequence of the feature from the
molecule. In the Text Pane or Feature List, right-click on a
feature and select Remove Feature. The feature information will be
removed from the molecule file, but the sequence will remain
unchanged. To undo a feature deletion, right-click in any pane in
the Molecule Viewer and select Undo. To remove the sequence of a
feature, see Inserting and Deleting Sequences. Marking Features You
can mark features in the Sequence and Graphics Panes and combine
them into new features.
Molecule Properties
[0316] The Molecule Properties dialog contains basic information
about a DNA/RNA or protein molecule, including a description,
references, associated genes, whether the molecule is circular or
linear, and database links. Information entered in this dialog is
shown in the Text Pane of the Molecule Viewer. To open the dialog,
click on the Molecule Properties button on the main toolbar or
select Properties from the Edit menu. The dialog is divided into
several tabs.
[0317] The General Tab includes database information about the
molecule file, including the database ID number and creation
date.
Molecule Tab
[0318] DNA/RNA molecules: Select Circular or Linear and DNA or RNA
from the dropdown lists.
[0319] Molecules with compatible overhangs will not circularize by
joining the overhangs; rather, the ends will be filled in. Only DNA
molecules flagged as Linear in this dialog can be used in the
Molecule Construction workspace as inserts or vectors. The user may
enter a brief description of the molecule in this field.
[0320] For Associated Genes, the user can click on Add Gene to add
a gene associated with the molecule to the list. A gene entry is
created in the table. Click in the editable text field to enter the
gene name. This creates a database link that is useful if you
export the molecule file to another format (GenBank, SWISSPROT,
VectorNTI, etc.). To delete a gene entry, click on it in the table,
then click on Remove Gene.
The Comment Tab Enter any comments about the molecule in this
field.
Standard Fields
[0321] This tab contains two subtabs. DNA/RNA molecules: The first
tab is called Division/Organella/Keywords. You can click in the
Division column and Organella column to select appropriate
categories for the molecule. These will be highlighted for the
molecule. Then you can enter the keywords as described below.
Keywords: Click on Add Keyword to add a database keyword associated
with the molecule to the list. A keyword entry is created in the
table. Click in the editable text field (DO) to enter the keyword.
This creates a database link that is useful if you export the
molecule file to another format. To delete a keyword, click on it
in the table, then click on Remove Keyword.
[0322] Source Organism: Click on this tab to display a table of
organisms associated with the molecule. Click on Add Organism to
add an organism associated with the molecule to the table. An
organism entry is created in both columns in the table. Click in
the editable text field in each column to enter the organism name
in Latin and English. This creates a database link that is useful
if you export the molecule file to another format (GenBank,
SWISSPROT, VectorNTI, etc.). To delete a source organism, click on
it in the table, then click on Remove Organism.
References Tab
[0323] Enter any references for the molecule in the field under
this tab. This is a simple text-entry field. If you want to export
the molecule in a particular format (e.g., GenBank), be sure to
enter text in that format.
[0324] Feature List
[0325] The Feature List is list of the defined features in the
molecule in an easy-to-read table format. The Feature List is
displayed in the Sequence Pane. Click on the Feature List button to
the left of the Sequence Pane to view the Feature List. Click on
the Sequence Pane button to return to a view of the sequence. Click
on a column header in the Feature List table to sort the list by
that column. Right-click on a feature in the list and select Edit
Feature Properties to open the Add/Edit Feature dialog. Right-click
on a feature in the list and select Copy Text to copy the feature
information to the computer clipboard in a tab-delimited format.
Right-click on a feature in the list and select Open Link to access
a variety of links to online databases with information about the
feature. Note that links are available for only certain types of
imported molecules.
Selecting a Sequence
[0326] Nucleotide and amino acid sequences are displayed in the
Sequence Pane of the Molecule Viewer. In the Viewer, you can select
part or all of a sequence, copy it, flag it as a feature, and
otherwise analyze it.
Selecting Part or All of a Sequence
[0327] There are a number of ways to select part or all of a
sequence. In the Molecule Viewer with the molecule displayed: Drag
your cursor in the Sequence Pane or Graphics Pane. The selected
part of the sequence will appear highlighted in both panes. Click
on a defined feature, ORF, or restriction site in the Graphics or
Text Pane, or double-click on a defined feature in the Feature
List. The sequence of that feature will appear selected in the
Sequence Pane. From the View menu or main toolbar, select Set
Selection. In the Set Selection dialog, define the selection area
and click on OK. The defined area will appear selected in the
Graphics and Sequence Panes. Right-click in the Sequence Pane and
select Select All to select the entire sequence.
Displaying Only the Selected Part of a Sequence
[0328] You can filter the display to show only the selected portion
of the sequence. With the selection made, go to the View menu and
select View Selection or click on the View Selection button on the
main toolbar. To return to a full view of the molecule, go to the
View menu and select View Entire Molecule or click on the View
Entire Molecule button on the main toolbar.
Finding a Sequence
[0329] To find a molecule sequence within a larger sequence,
right-click in the Sequence Pane in the Molecule Viewer and select
Find Sequence. In the, dialog, type or paste the sequence you want
to find, specify the search direction (Up or Down), and click on
Find Next. Click on Find Next again to find the next occurrence of
the sequence within the larger sequence. Click on Close to close
the dialog.
Inserting and Deleting Sequences DNA and Protein Molecules
[0330] You can insert a new DNA or protein sequence into an
existing DNA or protein molecule in the Molecule Viewer. Note that
this command will only insert a new sequence at the insertion
point; it will not overwrite any part of the existing sequence.
With the molecule displayed, locate the point in the sequence where
you want to insert the new sequence. Click on that point in the
Sequence Pane. From the Edit menu or main toolbar, select Insert
Sequence. The Insert Sequence dialog will open. In the dialog, note
the insertion point listed below the field. Type or paste the new
sequence into the field and click on OK. Note: Use only standard
code letters when entering the sequence. Nonstandard characters
will be marked with a ? in the Insert Sequence dialog and you will
be prompted to remove them before adding the new sequence. If you
are adding the sequence within a defined feature, the Feature Map
is Updated dialog will open, listing the features in the molecule
that will be affected by the insertion. In this dialog you can
remove any or all of the defined features that will be changed.
Note that this will not alter the change that you are making to the
sequence; it will only remove the defined feature(s) affected by
the change. Click on OK to make the changes. The sequence will be
added to the molecule. If you flagged a feature for deletion in the
Feature Map is Updated dialog, that feature will be removed.
[0331] To delete part of a sequence in the Molecule Viewer: With
the molecule displayed, drag the cursor in the sequence or Graphics
Pane to select the part of the sequence that you want to delete.
From the View menu or main toolbar, select Delete Sequence. Note:
You cannot delete the entire sequence in the Molecule Viewer. If
you are deleting the sequence within a defined feature, the Feature
Map is Updated dialog will open, listing the features in the
molecule that will be affected by the deletion. In this dialog you
can remove any or all of the defined features that will be changed.
Note that this will not alter the change that you are making to the
sequence; it will only remove the defined feature(s) affected by
the change. Click on OK to make the changes. The sequence will be
deleted from the molecule. If you flagged a feature for deletion in
the Feature Map is Updated dialog, that feature will be
removed.
[0332] For primers, the user can type, paste, and delete sequences
directly in the Sequence field of the Edit Primer Properties
dialog.
Copying a Sequence
[0333] You can copy a selected sequence to the computer clipboard.
In the Sequence Pane of the Molecule Viewer: Select the sequence.
Right-click on the selected sequence in the sequence pane and
select Copy.
[0334] The sequence will be copied to the computer clipboard. You
can then paste the copied sequence into your application of
choice.
Marking a Sequence
[0335] You can mark regions of interest in a DNA or protein
sequence with shading for easy comparison and reference. You can
also mark multiple regions (e.g., the exons of a gene of interest)
and label them as a single multi-segmented feature. In the Sequence
Pane or Graphics Pane of the Molecule Viewer, select the region you
want to mark, or click on the feature, ORF, or other defined
element that you want to mark. Click on the Mark Selection button
on the main toolbar, or select the command from the View menu or
context menu (if you right-click in the Graphics Pane). The
selected region will appear shaded-in the Sequence and Graphics
Panes. Repeat the steps above to mark multiple regions in the
sequence. You can then label them as a feature.
[0336] To unmark the sequence region: Select the marked region in
the Sequence or Graphic Pane; Click on the Unmark Selection button
on the main toolbar, or select the command from the View menu or
context menu (if you right-click in the Graphics Pane); and Click
on Unmark All to remove all the marks in the sequence.
Sequence Translation
[0337] You can use Vector Designer to translate the nucleotide
sequence in a DNA molecule into amino acids. Note that only the
Standard Genetic Code is available for translation. In the Molecule
Viewer with a nucleotide sequence displayed: Select the part of the
sequence that you want to translate. To select the entire sequence,
right-click in the Sequence Pane and select Select AH. To translate
the direct strand, click on the Translate Direct button on the main
toolbar, or select the command from View>Translation menu. To
translate the complementary strand, click on the Translate
Complementary button on the main toolbar, or select the command
from View>Translation menu. The translation will appear in the
Sequence Pane as amino acid codes above the nucleotide sequence. To
toggle between single-letter and three-letter amino acid codes,
click on the 1 Letter/3 Letter Code button from the main toolbar or
select the command from the View>Translation menu. To clear the
translation from the display, click on the Clear Translations
button on the main toolbar or select the command from the
View>Translation menu.
Designing Primers and PCR Products Designing Primers
[0338] You can use VectorDesigner to design primers for a target
sequence, or you can search for existing primers that are
compatible with the sequence. The resulting PCR products can then
be used in a variety of applications, including TOPO.RTM. Cloning,
Gateway.RTM. Cloning, and standard PCR analysis or molecule
construction. If you want to search for existing primers, the
primers must be saved in the Primers folder of the database as
separate primer files. The primer design settings are located in
the PCR Analysis dialog of the Molecule Viewer.
[0339] In the dialog, you specify the parameters for designing or
selecting the primers. Then VectorDesigner identifies one or more
primer designs. You can then: Save the primer designs with the
molecule or as separate files. Order the primers direct from
Invitrogen. Save the PCR product generated by the primers as a
separate molecule for further analysis. Evaluate the PCR product in
a cloning or molecule construction strategy.
[0340] To identify primers for a molecule sequence: In the Molecule
Viewer, select the region of the molecule for which you want to
design primers. Alternatively, if you are searching for existing
primers that are compatible with the molecule, you do not have to
select any region (available for TOPO.RTM. Cloning and molecule
construction applications only). Go to the Cloning menu, select the
appropriate subfolder for your application--TOPO Cloning, Gateway
Cloning, or Molecule Construction. Select Design Primers to Amplify
Selection to design primers for the selected sequence, or Find
Amplicon in Sequence Using Existing Primers to evaluate existing
primers for use with the molecule or selected sequence (available
on the TOPO Cloning and Molecule Construction submenus only). The
PCR Analysis dialog will open. The default values and available
options will differ slightly depending on the application you
selected (these differences are noted in the steps below). Under
the Primer Definition and Construction tab, the From and To fields
define the region that will be analyzed for primer designs. You can
change the numbers in these fields. Next, enter the primer design
parameters, or select the folders containing the saved primers that
you want to evaluate for compatibility with the molecule
sequence.
[0341] The following fields are only available if you selected
Design Primers to Amplify Selection: To include primer design
regions before and after the target sequence, enter a number of
bases in the Before and After fields. Maximum # of Outputs: Enter
the maximum number of primer pair designs to generate. Note that
VectorDesigner may generate fewer designs if no more can be found.
Tm: Enter the limits in degrees Celsius for primer melting
temperature (Tm) (temperature at which 50% of primer is a duplex)
in the Minimum and Maximum fields. Designs with Tm's outside this
range will be excluded. % GC: Enter the maximum and minimum percent
GC content for the primers in the fields. Designs with a percent GC
content outside this range will be excluded. (The percent GC of any
extensions are ignored.) Length: Enter the maximum and minimum
length (in bases) of each primer in the fields. Designs that fall
outside this range will be excluded. Nucleotide sequences such as
RENs attached to a primer's 5' end are included when calculating
primer length. Exclude Primers with Ambiguous Nucleotides: If your
sequence includes ambiguous bases (i.e., code letters other than A,
G, C, T), select this checkbox to exclude regions containing these
bases from the primer design search.
[0342] The following fields are only available if you selected Find
Amplicon in Sequence Using Existing Primers: Click on the Direct
button to select the folder containing the direct primer sequences
that you want to evaluate, and click on Complementary to select the
folder containing the complementary primer sequences to evaluate.
The Browse to Primer Folder dialog will open when you click on each
button. Select the folder and click on OK. The primers must be
saved in the Primers folder or subfolders as separate primer files.
Enter a percentage similarity in the Similarity>=Threshold
field. Each primer sequence must be at least this similar to the
molecule sequence to be selected by the designer. Select the
checkbox next to Last Nucleotides Must Have 100% Similarity to
specify a number of nucleotides at the 3' end of each primer that
must be 100% similar to the target sequence. Enter a number of
nucleotides in the field. Next, select the conditions of the PCR
reaction you are performing. If you are unsure of these values, use
the default values: Salt cone: The salt concentration of the PCR
reaction, in mMol. If you are unsure, use the default value of
50.0. Probe cone: The final concentration of the template in the
reaction, in pMol. If you are unsure, use the default value of
250.0. dG temp: The temperature of the free energy value of the
reaction, in degrees Celsius. If you are unsure, use the default
value of 25.0.
[0343] Under Cloning Termini, select the type of PCR product you
are generating. The available options will vary depending on your
cloning application. Click on an application below for more
information on how the primer and/or PCR product will be modified
based on your selection: TOPO.RTM. Cloning PCR Products;
Gateway.RTM. Cloning PCR Products; Molecule Construction PCR
Products.
[0344] For cloning applications, under Cloning strand, select the
strand whose sequence will be expressed: Direct or Complementary.
Note that this will affect the primer strand to which Directional
TOPO.RTM., Gateway.RTM., and other primer additions are added.
[0345] Next, select any sequence additions to each primer. This is
optional. Primer additions (such as RENs) can be used to add
sequences to the final PCR product for downstream applications such
as restriction-ligation and protein expression. Click on the Browse
button next to the Direct and/or Complementary fields. The Choose
Direct/Complementary Strand Addition dialog will open. Select the
strand additions in the dialog and click on OK. The additions will
be listed in the appropriate field. Additions to the primer
sequence will not be used in calculations of primer Tm, % GC, etc.
If you change the Cloning Strand (step 5 above) after selecting the
primer additions, the additions will switch to the other
strand.
[0346] Click on the Pairing, Structure and Uniqueness tab to access
additional primer specifications. Max. Tm Difference: Specify the
maximum difference in melting temperature between sense and
antisense primers in degrees Celsius. Max. % GC Difference: Specify
the maximum percentage difference in GC content between sense and
antisense primers. Note the differences in GC content between the
two primer regions of the sequence when specifying this difference;
a difference that is too small may result in no primers being
found. Primer-Primer Complementarity: Permitted with dG>=:
Select this checkbox and enter the minimum permitted value for free
energy of a primer-primer duplex. Primer pairs which have a free
energy value>/=to this number will be accepted. Primer-Primer
Complementarity: 3' End Permitted with dG>=: Select this
checkbox and enter the minimum permitted value for free energy of
complementarity between the 3'-end of the primers (the final 5
bases of each primer will be evaluated). Primer pairs which have a
3'-end complementarity free energy value>/=to this number will
be accepted. Exclude Primers With: In the Repeat field, enter the
maximum number of base-pair repeats allowed in each primer. In the
Palindrome field, enter the maximum permitted length of palindromes
in each primer sequence. In the Hairpin Loops field, enter the
minimum permitted value for free energy of hairpin loops within
each primer. Primer Uniqueness: Select this checkbox to reject
primers above a certain percentage similarity to secondary sites
within either the entire sequence or within the amplicon. Enter an
percentage similarity in the field, and select
Within Entire Sequence or within Amplicon Only.
[0347] Click on OK to design the primers. You will be prompted to
send the PCR product for the first (highest ranked) primer pair
directly to the appropriate molecule construction workspace as an
insert. If you click on No, all the primer pairs generated will be
added to the PCR Primers folder in the Text Pane of the Molecule
Viewer.
Primer Designs and PCR Products
[0348] After you have designed primers from a molecule sequence
using the tools in the Molecule Viewer, the primer designs and
their PCR products will be listed in the PCR Primers folder of the
Text Pane.
Viewing Primer Designs in the Text Pane
[0349] In the Text Pane, information about each primer design is
included in the PCR Primers folder. Note: Only the designs for most
recent target sequence are saved in this folder. If you design
primers for a different target sequence, the new designs will
replace any old ones. To preserve the primer designs for a
particular target sequence, save them as features as described
below.
[0350] In the PCR Primers folder, the PCR product for each primer
pair has its own subfolder: Double-click on the subfolder to select
the amplicon region in the graphics and Sequence Panes; Click on
the +next to the PCR product folder name view the information for
the primer pair; Double-click on each individual primer sequence in
the PCR product folder to highlight that sequence in the graphics
and Sequence Panes.
Ordering Primers from Invitrogen
[0351] Click on the Order from Invitrogen link next to each primer
in the Text Pane to order the primer from Invitrogen. You will be
prompted to enter a primer name, and the primer sequence will
automatically be loaded into Invitrogen's online ordering system.
You can specify the details of your order (purity, synthesis scale,
etc.) on the Web site.
Adding a PCR Product to a Workspace
[0352] To load a PCR product in the TOPO.RTM. Cloning, Gateway.RTM.
Cloning, or Molecule Construction workspace as an insert: In the
PCR Primers folder in the Text Pane, right-click on the Product
folder for the PCR product and select Add PCR Product to
<application>Workspace (Note that specific application
workspace listed will depend on which type of PCR analysis was used
to generate the product); and the workspace will be displayed in
the main VectorDesigner window, and the PCR product will be listed
in the Insert field.
Saving the PCR Product as a New Molecule
[0353] To open the PCR product as a separate molecule: Right-click
on the PCR product folder in the PCR Primers folder and select Open
PCR Product in New Molecule Viewer. A new Molecule Viewer will open
displaying the amplicon sequence as a linear DNA molecule, with the
primers marked as features. Note that the new molecule is not
automatically saved in the database; use the Save command in the
Viewer to save the new molecule.
Saving Individual Primer Designs as New Molecules
[0354] To save the primer designs as individual primers in the
database: Right-click on the primer sequence in the Text Pane
(i.e., the actual sequence of the specific direct or complementary
primer) and select Save Primer into DB. The Save As dialog box will
open, prompting you to specify the primer name. Primers may be
saved in the Primers folder or subfolders. To open the new primer
file, go to the Database Browser window and double-click on the
primer in the Primers folder. The Edit Primer Properties dialog
will open.
Adding a PCR Product to the Feature Map
[0355] To add one more more PCR products to the Feature Map: In the
PCR Primers folder in the Text Pane, right-click on a Product
folder and select Annotate Analysis Item. In the Add/Edit Feature
dialog; fill out the information and click on OK to add the PCR
product to the feature map. To undo this command, right-click in
the Text Pane and select Undo Annotate Analysis. Adding Primer
Designs to the Feature Map
[0356] To add all primer designs to the Feature Map: In the Text
Pane, right-click on the PCR Primers folder and select Annotate
Analysis. The Annotate Analysis dialog will open; select the
feature type and enter a feature name and description, and click on
OK. Note that you can only fill out a single name all the primer
designs; the individual primers will be given the name plus a
numerical extension (<primer name>.sub.-1, <primer
name>.sub.-2, etc.).
[0357] To add one or more primer pairs to the Feature Map: In the
PCR Primers folder in the Text Pane, right-click on the Product
folder for a primer pair, or hold down the Control key and click on
multiple Product folders to select them and right-click on the
selection. Select Annotate Analysis from the context menu. The
Annotate Analysis dialog will open; select the feature type and
enter a feature name and description, and click on OK. Note that
you can only fill out a single name all the primer designs; the
individual primers will be given the name plus a numerical
extension (<primer name>1, <primer name>.sub.-2, etc.).
To add an individual primer sequence to the Feature Map: In the PCR
Primers folder in the Text Pane, open the Product folder for the
design you want and right-click on the primer sequence (i.e., the
actual sequence of the specific direct or complementary primer).
Select Annotate Analysis from the context menu. The Annotate
Analysis dialog will open; fill out the information and click on
OK. The primer sequence will be added. To undo these commands,
right-click in the Text Pane and select Undo Annotate Analysis.
Deleting Primer Designs
[0358] To delete a PCR primer design from the molecule file: In the
PCR Primers folder in the Text Pane, right-click on the Product
folder and select Remove Site. The information for the primer
designs and PCR product will be removed. (Note that the actual
molecule sequence will not be affected.) To remove all primer
designs from the molecule, right-click on the PCR Primers folder in
the Text Pane and select Remove Analysis.
Marking/Highlighting Primer Designs
[0359] To mark a single PCR product in the Sequence and Graphics
Panes with shading: In the PCR Primers folder in the Text Pane,
right-click on the Product folder and select Mark Site.
[0360] To mark multiple PCR products in the Sequence and Graphics
Panes with shading: In the PCR Primers folder in the Text Pane,
hold down the Control key and click the Product folders to
select-them, then right-click on the selection and select Mark
Selected Items.
[0361] To mark a single primer sequence in the Sequence and
Graphics Panes with shading: In the PCR Primers folder in the Text
Pane, open the Product folder for the design, right-click on the
specific primer sequence, and select Mark Site. (Note that you must
right-click on the actual primer sequence, not the primer
name.)
[0362] To mark multiple primer sequences in the Sequence and
Graphics Panes with shading: In the PCR Primers folder in the Text
Pane, open the Product folder(s) containing the primer designs,
hold down the Control key and click on the primer sequences to
select them, and then right-click on the selection and select Mark
Selected Items. (Note that you must select the actual primer
sequences, not the primer names.) To undo a marked region, select
the PCR product and select View>Unmark Selection.
[0363] ORFs and Restriction Mapping Open Reading Frames
[0364] You can identify open reading frames (ORFs) in DNA molecules
using the ORF Search tool in the Molecule Viewer. ORFs identified
by the tool are shown in the Graphics, Sequence, and Text Panes, as
described below.
Identifying ORFs
[0365] Using the ORF Search tool, you can set the minimum ORF size,
the start and stop codons to search for, and other parameters, and
VectorDesigner will generate a list of defined ORFs. To perform the
ORF search: In the Molecule Viewer displaying a DNA molecule, click
on the ORF Search button or go to the Tools menu and select ORF
Search. In the ORF Search dialog, specify the Minimum ORF Size (in
codons) and select the Nested ORFs checkbox if you want to search
for nested ORFS (ORFs that have the same stop codon but different
start codons). In Start Codons and Stop Codons fields, enter one or
more start and stop codons to search for when identifying ORFs.
Separate each codon by a space. To reset the fields, click on Reset
to Default. Select Include Stop Codon in ORF if you want the stop
codon to be considered part of the ORF. Otherwise, the stop codon
will not be included in each ORF defined in the sequence. Click on
OK to search for the ORFs.
[0366] The ORFs will be marked on the sequence in the Graphics and
Sequence Panes, and a folder called Open Reading Frames will be
created in the Text Pane. If you perform the ORF search again, the
existing search results will be overwritten.
Viewing and Selecting ORFs
[0367] Each pane in the Molecule Viewer has different tools for
viewing and selecting ORFs. Graphics Pane: In the Graphics Pane,
ORFs are marked by thin directional arrows aligned with the
sequence. Hold your cursor over an ORF arrow to display a popup
information box for that ORF. Click on an arrow to highlight the
ORF in the Sequence Pane. Right-click on an ORF and select Find in
Tree to select the ORF in the Text Pane.
[0368] Sequence Pane: In the Sequence Pane, ORFs are marked by
black bars above the sequence. Click on an ORF arrow in the
Graphics Pane or an ORF name in the Text Pane to highlight the
sequence in the Sequence Pane.
[0369] Text Pane: In the Text Pane, information about identified
ORFs is included in a folder called Open Reading Frames. In this
folder, each ORF is listed by its position in the sequence. The
notation (D1, D2, D3, or C1, C2, C3) refers to the strand
containing the ORF and its reading frame in the molecule sequence.
For example, in a direct strand sequence beginning ATGTGTACTCCTTA .
. . (SEQ ID NO:9), an ORF beginning with ATG would have the
notation D1 and an ORF beginning with GTG would have the notation
D3. Double-click on an ORF name in the folder to highlight the ORF
in the Graphics and Sequence Panes. Click on the +next to each ORF
name to view the start codon, stop codon, region of the sequence,
and length of each ORF.
Adding ORFs to the Feature Map
[0370] You can add ORFs to the Feature Map in one of two ways: In
the Text Pane, right-click on an ORF in the Open Reading Frames
folder and select Annotate Analysis. The Annotate Analysis dialog
will open; fill out the information and click on OK to add the ORF
to the feature map. Note that this dialog will only enable you to
add the ORF sequence as defined by the ORF Search tool. To undo
this command, right-click in the Text Pane and select Undo Annotate
Analysis. If you want to alter the start and/or endpoint of the ORF
before defining it as a feature, right-click on the ORF in the Open
Reading Frames folder and select Annotate Analysis Item. This will
open the Add/Edit Feature dialog, in which you can change the
start/endpoint of the feature. To undo this command, right-click in
the Text Pane and select Undo Annotate Analysis.
Deleting ORFs
[0371] You can delete an ORF definition and information without
removing the ORF sequence from the molecule. In the Text Pane,
right-click on an ORF and select Remove Site. The ORF information
will be removed from the panes, but the sequence will remain
unchanged. To remove all ORF definitions from the molecule,
right-click on the Open Reading Frames folder in the Text Pane and
select Remove Analysis. To undo an ORF deletion, right-click in any
pane in the Molecule Viewer and select Undo. To remove the sequence
of an ORF, see Inserting and Deleting Sequences.
Marking/Highlighting ORFs
[0372] You can mark the ORF sequence in the Sequence and Graphics
Panes with shading. In the Text Pane, right-click on the ORF and
select Mark Site. In the Graphics Pane, right-click on the ORF
arrow and select Mark Selection. Or, with the ORF selected in the
Sequence Pane, go to the View menu and select Mark Selection. To
undo a marked ORF, select the ORF and select View>Unmark
Selection. Restriction Analysis
[0373] Vector Designer can identify the restriction enzyme cut
sites in a DNA molecule using a built-in database of restriction
enzymes. You can use the cut sites to generate restriction
fragments for molecule construction.
Restriction Map Search
[0374] To perform restriction analysis: In the Molecule Viewer
displaying a DNA molecule, click on the Restriction Map Search
button (RMap) or go the Tools menu and select Restriction Map
Search. In the Restriction Map Search dialog, select the category
of enzymes that you want to use from the Use Enzymes list:
Frequently Used Enzymes have been identified by Invitrogen. Click
here for a list. 7+Cutters, 6 Cutters, 5 Cutters, etc. refer to the
number of base pairs in the recognition site of each enzyme.
Enzymes in the 5' Overhang category result in fragments with a 5'
overhang; enzymes in the 3' Overhang category result in fragments
with a 3' overhang. If you select Customized, click on the
Customize button to select the particular enzymes you want to use.
The Enzymes List dialog will open.
[0375] Next, enter a number in the Display Enzymes with
<=Recognition Sites field. The Designer will analyze the
sequence and use only those enzymes with less than or equal to that
number of cut sites. Alternatively, select Unlimited to not filter
the enzyme list by number of cut sites. When you have made your
selections, click on OK.
Viewing and Selecting Restriction Sites
[0376] Each pane in the Molecule Viewer has different tools for
viewing and selecting restriction sites. Graphics Pane: In the
Graphics Pane, restriction sites are marked by blue-green lines
from the site to the name of the restriction enzyme. Hold your
cursor over the restriction enzyme name to display a popup
information box for that site. Click on a restriction site to
highlight the site in the Sequence Pane. Right-click on a
restriction site and select Find in Tree to select the site in the
Text Pane. See Restriction Fragments for instructions on selecting
fragments in the Graphics Pane.
[0377] Sequence Pane: In the Sequence Pane, restriction sites are
marked by blue bars above the sequence and the name of the enzyme
above the bar. Click on the blue bar above the sequence to display
a line through the sequence showing the exact cut site and overhang
created by the enzyme. See Restriction Fragments for instructions
on selecting fragments in the Sequence Pane.
[0378] Text Pane: In the Text Pane, information about identified
restriction sites is included in a folder called Restriction Map.
In this folder, each restriction site is listed by enzyme name.
Double-click on a restriction site in the folder to highlight the
site in the Graphics and Sequence Panes. Click on the +next to each
enzyme name to view the complete name of the organism and the
locations in the sequence where it cuts. Click on the Order from
Invitrogen link to order the restriction endonuclease from
Invitrogen. You will be linked to Invitrogen's online catalog page
for the enzyme.
Adding Restriction Sites to the Feature Map
[0379] You can add restriction sites to the Feature Map. In the
Text Pane, right-click on a restriction site in the Restriction Map
folder and select Annotate Analysis. The Annotate Analysis dialog
will open; fill out the information and click on OK to add the
restriction site. To undo this command, right-click in the Text
Pane and select Undo Annotate Analysis. To remove all restriction
site definitions from the molecule, right-click on the Restriction
Map folder in the Text Pane and select Remove Analysis.
Restriction Fragments Selecting a Restriction Fragment
[0380] You can select the region between two restriction enzyme cut
sites in the Graphics or Sequence Pane to generate a restriction
fragment. See Restriction Analysis for information on generating a
list of cut sites. Before proceeding, you may want to limit the
display to just the enzymes you are interested in using the Link
Mode feature.
[0381] In the Graphics Pane with the restriction enzyme cut sites
displayed, click on a restriction enzyme name, hold down the Shift
key, and click on a second enzyme name. The region between the two
cut sites will appear selected in the Sequence Pane.
[0382] In the Sequence Pane with the restriction enzyme cut sites
displayed, click on the blue bar above a restriction site, hold
down the Shift key, and click on a second blue bar. The region
between the two cut sites will appear selected. Now you can copy
the selected fragment, define it as a feature, or add it to the
Molecule Construction workspace as an insert or a vector.
[0383] Adding a Fragment to the Molecule Construction Workspace
[0384] To add the feature to the Molecule Construction workspace as
an insert or a vector: With the fragment selected, go to the
Cloning>Molecule Construction menu and select Add Restriction
Fragment to Workspace as an Insert or Add Restriction Fragment to
Workspace as a Vector. The Molecule Construction workspace will be
displayed in the main VectorDesigner window, and the fragment will
be listed in the appropriate field (Insert or Vector).
Cloning Tools Molecule Construction
[0385] VectorDesigner provides automated tools for in silico
construction of DNA molecules (e.g., expression clones) from
existing sequences based on conventional cloning methodologies
(e.g., restriction-ligation, TA cloning). VectorDesigner also
provides tools for in silico molecule construction using
Gateway.RTM. Cloning and TOPO.RTM. Cloning technologies.
[0386] Using these tools, you first design and select the
sequences/molecules that you want to use to create the new molecule
and add them to the Molecule Construction workspace. When you click
on Clone, VectorDesigner will automatically select the optimal
sites for recombination and generate and display the new design.
The tools for in silico molecule construction are located in the
Molecule Construction workspace in the main VectorDesigner window.
Click on the Molecule Construction tab to view the workspace.
[0387] To construct molecules, you must first design and/or select
an insert sequence and a vector sequence, as described below. The
insert and vector sequences must have compatible ends (e.g., blunt
ends or compatible overhangs). VectorDesigner can be used to
construct a DNA molecule from one insert and one vector at a time.
Vector NTI.RTM. software provides a suite of additional tools and
options for constructing molecules.
Selecting Inserts
[0388] Inserts must be linear DNA sequences, and must have
compatible ends with the vector you select. Examples include the
following: Restriction fragments--see Restriction Analysis and
Restriction Fragments; PCR products--see Designing Primers and
Primer Designs and PCR Products; Linear DNA molecules (blunt-ended
or with T-A extensions)
[0389] Selecting Inserts in the Molecule Construction Workspace
[0390] If the insert has been saved as a molecule in the
VectorDesigner database, you can select it in the Molecule
Construction workspace. Note that the insert must be saved in the
DNA/RNAs folder or a subfolder. Click on the Browse in Insert
button in the workspace. The window will expand, displaying
navigation tools at the bottom. Using the folder tree in the
left-hand part of the window, navigate to the folder containing
your insert. Click on the insert name in the right-hand part of the
window. The insert will be added to the Insert field in the
workspace. (Note that you may need to scroll up in the window to
view the Insert field.) Selecting Inserts in the Molecule
Viewer
[0391] The Molecule Viewer includes a number of tools for
generating inserts and transferring them to the Molecule
Construction workspace. See Restriction Fragments for instructions
on selecting a restriction fragment in the Molecule Viewer and
adding it to the Molecule Construction workspace as an insert. See
Primer Designs and PCR Products, for instructions on selecting a
PCR product in the Molecule Viewer and adding it to the Molecule
Construction workspace as an insert.
[0392] When you design primers for a molecule sequence using the
tools on the Cloning>Molecule Construction menu, you will be
prompted to send the PCR product from the primary design directly
to the Molecule Construction workspace. (See Designing Primers.) If
you select No, the product will be added to the PCR Primers folder
in the Text Pane and you can add it to the workspace from there.
(See Primer Designs and PCR Products). You can transfer the entire
molecule to the workspace as an insert. In the Molecule Viewer, go
to the Cloning>Molecule Construction menu and select Add Entire
Molecule to Workspace as Insert. Note that the molecule must be
linear for this command to be available. When you use any of the
methods above, the Molecule Construction workspace window will be
displayed and the selected sequence will be listed in the Insert
field.
Selecting Vectors
[0393] Vectors must be linear DNA sequences, and must have
compatible ends with the insert you select. Examples include the
following: Restriction fragments--see Restriction Analysis and
Restriction Fragments; Linear DNA molecules (e.g., linearized
vectors; blunt-ended or with T-A extensions) Selecting Vectors in
the Molecule Construction Workspace
[0394] If the vector has been saved as a molecule in the
VectorDesigner database, you can select it in the Molecule
Construction workspace. Click on the Browse in Vector button in the
workspace. The window will expand, displaying navigation tools at
the bottom. Using the folder tree in the left-hand part of the
window, navigate to the folder containing your vector. Click on the
vector name in the right-hand part of the window. The vector will
be added to the Vector field in the workspace. (Note that you may
need to scroll up in the window to view the Insert field.)
Selecting Vectors in the Molecule Viewer
[0395] The Molecule Viewer includes tools for generating
restriction fragments that can used as vectors. You can also
transfer the entire molecule to the workspace as a vector. See
Restriction Fragments for instructions on selecting a restriction
fragment in the Molecule Viewer and adding it to the Molecule
Construction workspace as a vector. You can transfer the entire
molecule to the workspace as a vector. In the Molecule Viewer, go
to the Cloning>Molecule Construction menu and select Add Entire
Molecule to Workspace as Vector. Note that the molecule must be
linear for this command to be available. When you use either of
these methods, the Molecule Construction workspace window will be
displayed and the selected sequence will be listed in the Vector
field.
[0396] Incompatible Termini
[0397] If you have added inserts and/or vectors with incompatible
termini to the workspace, an alert message will appear in the
left-hand pane of the Molecule Construction workspace. You will be
prompted to: Select different inserts/vectors; Modify the
inserts/vectors using restriction enzymes that will result in
compatible termini; or Fill/trim any incompatible overhangs.
[0398] To modify an insert or vector, open it in the Molecule
Viewer and use the editing tools in the Viewer to make the changes.
Then re-add it to the Molecule Construction workspace. To fill or
trim incompatible overhangs, use the pulldown boxes in the Insert
and Vector fields in the Molecule Construction workspace to select
the appropriate options (None, Fill, or Trim).
Creating the New Molecule
[0399] When you have added a compatible insert to the Insert field
and a compatible vector to the Vector field, the Clone button in
the Molecule Construction workspace will become active. Click on
Clone to create the new molecule. The molecule will open in a new
Molecule Viewer window. The insert may clone into the vector in
both orientations, depending on the compatibility of the terminals.
In this case, two new molecules will open. Use the Save command in
the Molecule Viewer to save the new molecule.
[0400] Information about the New Molecule
[0401] Any features from the constituent molecules will be
preserved in the new molecule, except for features that may be
eliminated or truncated in the reaction. In addition to the
standard information provided in the Molecule Viewer, the following
information is provided for constructed molecules: In the Text
Pane, the Design Description outlines the steps for the appropriate
cloning reaction. In the Text Pane, the Component Fragments folder
provides a description of each molecule fragment used to construct
the molecule. Under each fragment, click on Open in Molecule Viewer
to open the fragment in a new Viewer window (note that the fragment
in the new Viewer window will not be saved).
[0402] Analysis of the New Molecule
[0403] You can now analyze the new molecule using analysis tools
such as the open reading frame and sequence translation tools in
VectorDesigner to verify that the DNA sequence is inserted and will
be expressed as intended.
[0404] Gateway Cloning Overview of Gateway.RTM. Cloning
[0405] Gateway.RTM. Technology is based on the bacteriophage lambda
site-specific recombination system
(atth.times.attR<=>attB.times.attP), which involves DNA
recombination sequences {att sites) and proteins that bring
together the target sites, cleave them, and covalently attach the
DNA. Gateway.RTM. Technology uses lambda recombination to
facilitate the transfer of heterologous DNA sequences (flanked by
modified att sites) between vectors. Two recombination reactions
constitute the basis of the Gateway.RTM. Technology: (1) BP
Reaction: Facilitates recombination of an attB substrate (attB-PCR
product or a linearized attB expression clone) with an att?
substrate (donor vector) to create an a/L-containing entry clone.
This reaction is catalyzed by BP Clonase.TM. II enzyme mix; and (2)
LR Reaction: Facilitates recombination of an attL substrate (entry
clone) with an attR substrate (destination vector) to create an
aftB-containing expression clone (see diagram below). This reaction
is catalyzed by LR Clonase.TM. II enzyme mix.
[0406] More information about Gateway.RTM. Technology: Gateway.RTM.
Technology can be found in the Gateway.RTM. Technology manual,
which is available on the World Wide Web at invitrogen.com.
Gateway.RTM. Cloning
[0407] VectorDesigner provides automated tools for in silico
construction of Gateway.RTM. entry clones and Gateway.RTM.
expression clones from existing sequences and vectors. Using
VectorDesigner, you can construct a: Gateway.RTM. entry clone using
an atiB substrate (attB-PCR product or attB-expression clone) and a
donor vector (BP reaction); and/or Gateway.RTM. expression clone
using an entry clone (attL substrate) and destination vector (LR
reaction). In VectorDesigner, you first design and select the
substrates and vectors that you want to use to create the new entry
clone or expression clone and add them to the Gateway.RTM. Cloning
workspace. When you click on Clone, VectorDesigner will
automatically recombine the sequences and generate and display the
new molecule. The tools for in silico Gateway.RTM. Cloning are
located in the Gateway.RTM. Cloning workspace in the main
VectorDesigner window. Click on the Gateway.RTM. Cloning tab to
view the workspace.
[0408] To construct molecules, you must first design and/or select
an insert and a vector: The insert in VectorDesigner is an attB
substrate (attB-FCR product or attB-expression clone) if you are
generating a Gateway.RTM. entry clone (BP reaction) or an entry
clone if you are generating a Gateway.RTM. expression clone (LR
reaction). The vector in VectorDesigner is a Gateway.RTM. donor
vector if you are generating a Gateway.RTM. entry clone (BP
reaction) or a Gateway.RTM. destination vector if you are
generating a Gateway.RTM. expression clone (LR reaction). Files of
Gateway.RTM. vectors are included in the Invitrogen
Vectors>Gateway Vectors folder in VectorDesigner.
[0409] Selecting Inserts
[0410] The type of insert you select will depend on whether you
want to perform a BP reaction to generate a Gateway.RTM. entry
clone or an LR reaction to generate a Gateway.RTM. expression
clone.
[0411] To generate a Gateway.RTM. entry clone (BP reaction),
inserts can be: attB PCR products--see Designing Primers and Primer
Designs and PCR Products for generating and selecting
Gateway.RTM.-adapted PCR products containing attB sites; any DNA
molecule containing attB sites; a Gateway.RTM. expression
clone.
[0412] To generate a Gateway.RTM. expression clone (LR reaction),
the insert must be a Gateway.RTM. entry clone. You can generate an
entry clone using the following methods: Perform a BP reaction
using an attB substrate and a donor vector; Use TOPO.RTM. Cloning
or conventional cloning methods to insert your sequence of interest
into a pENTR/TOPO or pENTR vector from Invitrogen. Molecule files
of top-selling pENTR/TOPO vectors are provided in the Invitrogen
Vectors>TOPO Vectors>Directional folder, and files of
top-selling pENTR vectors are provided in the Invitrogen
Vectors>Gateway Vectors>pENTR Vectors folder in
VectorDesigner.
[0413] Selecting Inserts in the Gateway.RTM. Cloning Workspace
[0414] If the insert has been saved as a molecule in the
VectorDesigner database, you can select it in the Gateway.RTM.
Cloning workspace. Note that the insert must be saved in the
DNA/RNAs folder or a subfolder. Click on the Browse in Insert
button in the workspace. The window will expand, displaying
navigation tools at the bottom. Using the folder tree in the
left-hand part of the window, navigate to the folder containing
your insert. Click on the insert name in the right-hand part of the
window. The insert will be added to the Insert field in the
workspace. (Note that you may need to scroll up in the window to
view the Insert field.)
[0415] Selecting Inserts in the Molecule Viewer
[0416] The Molecule Viewer includes tools for designing
Gateway.RTM.-adapted PCR products and transferring them to the
Gateway.RTM. Cloning workspace. See Designing Primers. See Primer
Designs and PCR Products for instructions on selecting a PCR
product in the Molecule Viewer and adding it to the Gateway.RTM.
Cloning workspace as an insert. When you design primers for a
molecule sequence using the tools on the Cloning>Gateway Cloning
menu, you will be prompted to send the resulting attB PCR product
directly to the Gateway.RTM. Cloning workspace. You can transfer an
entire molecule to the workspace as an insert. In the Molecule
Viewer, go to the Cloning>Gateway Cloning menu and select Add
Molecule to Workspace as Insert. When you use any of the methods
above, the Gateway.RTM. Cloning workspace window will be displayed
and the selected sequence will be listed in the Insert field.
Selecting Vectors
[0417] The type of vector you select will depend on whether you
want to perform a BP reaction to generate a Gateway.RTM. entry
clone or an LR reaction to generate a Gateway.RTM. expression
clone: To generate a Gateway.RTM. entry clone (BP reaction), you
must select a Gateways donor vector. Molecule files of top-selling
donor vectors are provided in the Invitrogen Vectors>Gateway
Vectors>pDONR Vectors folder in VectorDesigner. Sequences of
additional donor vectors can be located by searching the Invitrogen
Vectors Web database. To generate a Gateway.RTM. expression clone
(LR reaction), you must select a Gateway.RTM. destination vector.
Molecule files of top-selling destination vectors are provided in
the Invitrogen Vectors>Gateway Vectors>pDEST Vectors folder
in VectorDesigner. Sequences of additional destination vectors can
be located by searching the Invitrogen Vectors Web database.
Selecting Vectors in the Gateway.RTM. Cloning Workspace
[0418] If the Gateway.RTM. vector is in the VectorDesigner
database, you can select it in the Gateway.RTM. Cloning workspace:
Click on the Browse in Vector button in the workspace. The window
will expand, displaying navigation tools at the bottom. Using the
folder tree in the left-hand part of the window, navigate to the
folder containing your vector. Click on the vector name in the
right-hand part of the window. The vector will be added to the
Vector field in the workspace. (Note that you may need to scroll up
in the window to view the Insert field.)
Selecting Vectors in the Molecule Viewer
[0419] From the Molecule Viewer, you can transfer the Gateway.RTM.
vector to the workspace. Go to the Cloning>Gateway Cloning menu
and select Add Molecule to Workspace as Vector. The Gateway.RTM.
Cloning workspace window will be displayed and the selected
sequence will be listed in the Vector field.
Creating the New Molecule
[0420] After you have added a compatible insert to the Insert field
and a compatible vector to the Vector field, the Clone button in
the Molecule Construction workspace will become active. If you
select incompatible inserts and/or vectors, an alert message will
appear in the left-hand pane of the Molecule Construction
workspace, and you will be prompted to select different
inserts/vectors.
[0421] Click on Clone to create the new molecule. The molecule will
open in a new Molecule Viewer window. Use the Save command in the
Molecule Viewer to save the new molecule. Information about the New
Molecule
[0422] Any features from the constituent molecules will be
preserved in the new molecule, except for features that are
eliminated and added in the recombination reaction (e.g., the atth
sites in an entry clone and attR sites in a destination vector will
be eliminated and replaced by attB sites in the expression
clone).
[0423] In addition to the standard information provided in the
Molecule Viewer, the following information is provided for
constructed molecules: In the Text Pane, the Design Description
outlines the steps for the appropriate cloning reaction. In the
Text Pane, the Component Fragments folder provides a description of
each molecule fragment used to construct the molecule. Under each
fragment, click on Open in Molecule Viewer to open the fragment in
a new Viewer window (note that the fragment in the new Viewer
window will not be saved). Analysis of the New Molecule
[0424] You can analyze entry clones and expression clones using the
open reading frame and sequence translation analysis tools in
VectorDesigner to verify that the sequence has the correct reading
frame and translation.
[0425] TOPO.RTM. Cloning TOPO.RTM. technology uses the unique
properties of vaccinia DNA topoisomerase I to mediate rapid,
joining of PCR products into plasmid vectors. No ligase, post-PCR
procedures, or PCR primers containing specific sequences are
required. For more information, visit the TOPO.RTM. Cloning Web
site on the World Wide Web at invitrogen.com.
[0426] Zero Blunt.RTM. TOPO.RTM. Cloning
[0427] Each Zero Blunt.RTM. TOPO.RTM. vector has Topoisomerase I
covalently bound to both vector terminals. This allows blunt-end
PCR products to ligate efficiently with the vector.
TOPO.RTM. TA Cloning.RTM.
[0428] Tag DNA polymerase has a nontemplate-dependent terminal
transferase activity that adds a single deoxyadenosine (A) to the
3' ends of PCR products. Each TOPO.RTM. TA vector has overhanging
3' deoxythymidine (T) residues and Topoisomerase I covalently bound
to the vector terminals. This allows PCR inserts generated with Taq
polymerase to ligate efficiently with the vector.
Directional TOPO.RTM. Cloning
[0429] In this system, PCR products are directionally cloned by
adding four bases to the forward primer (CACC). The
TOPO.RTM.-charged overhang in the cloning vector (GTGG) invades the
5' end of the PCR product, anneals to the added bases, and
stabilizes the PCR product in the correct orientation. Inserts can
be cloned in the correct orientation with efficiencies equal to or
greater than 90%.
[0430] TOPO.RTM. Cloning VectorDesigner provides automated tools
for in silico construction of expression clones from DNA sequences
using TOPO.RTM. cloning technology. You can construct clones using
TOPO.RTM. TA Cloning, Directional TOPO.RTM. Cloning, and Blunt
TOPO.RTM. Cloning methods.
[0431] In VectorDesigner, you first design and select the sequences
(typically PCR products) and TOPO.RTM. vectors that you want to use
to create the new expression clone and add them to the TOPO.RTM.
Cloning workspace. When you click on Clone, VectorDesigner will
automatically recombine the sequences and generate and display the
new molecule.
[0432] The tools for in silico TOPO.RTM. Cloning are located in the
TOPO.RTM. Cloning workspace in the main VectorDesigner window.
Click on the TOPO.RTM. Cloning tab to view the workspace.
[0433] To construct molecules, you must first design and/or select
an insert and a vector: The insert should be a DNA
sequence--typically a PCR product in TOPO.RTM.
applications--configured for the type of TOPO.RTM. Cloning you want
to perform (e.g., TA, Directional, Blunt). The vector should be an
appropriate TOPO.RTM. vector. Files of TOPO.RTM. vectors are
included in the Invitrogen Vectors>TOPO Vectors folder in
VectorDesigner.
Selecting Inserts
[0434] Inserts must be linear DNA sequences. They can be: PCR
products--see Designing Primers and Primer Designs and PCR Products
for generating and selecting TOPO.RTM.-adapted PCR products; Linear
DNA molecules--If you select a molecule with Blunt ends, use a Zero
Blunt.RTM. TOPO.RTM. Vector; with 3' A overhangs, use a TOPO.RTM.
TA Vector; or with a CACC sequence at one end, use a Directional
TOPO.RTM. Vector or Zero Blunt.RTM. TOPO.RTM. Vector.
Selecting Inserts in the TOPO.RTM. Cloning Workspace
[0435] If the insert has been saved as a molecule in the
VectorDesigner database, you can select it in the TOPO.RTM. Cloning
workspace. Note that the insert must be saved in the DNA/RNAs
folder or a subfolder. Click on the Browse in Insert button in the
workspace. The window will expand, displaying navigation tools at
the bottom. Using the folder tree in the left-hand part of the
window, navigate to the folder containing your insert. Click on the
insert name in the right-hand part of the window. The insert will
be added to the Insert field in the workspace. (Note that you may
need to scroll up in the window to view the Insert field.)
Selecting Inserts in the Molecule Viewer
[0436] The Molecule Viewer includes tools for designing
TOPO.RTM.-adapted PCR products and transferring them to the
TOPO.RTM. Cloning workspace. See Designing Primers. See Primer
Designs and PCR Products for instructions on selecting a PCR
product in the Molecule Viewer and adding it to the TOPO.RTM.
Cloning workspace as an insert.
[0437] When you design primers for a molecule sequence using the
tools on the Cloning>TOPO Cloning menu, you will be prompted to
send the resulting PCR product directly to the TOPO.RTM. Cloning
workspace. You can transfer an entire molecule to the workspace as
an insert. In the Molecule Viewer, go to the Cloning>TOPO
Cloning menu and select Add Molecule to Workspace as Insert. Note
that the molecule must be linear for this command to be available.
When you use any of the methods above, the TOPO.RTM. Cloning
workspace window will be displayed and the selected sequence will
be listed in the Insert field.
Selecting Vectors
[0438] Vectors must be linear TOPO.RTM. vectors, and must have
compatible ends with the insert you select. Molecule files of
top-selling TOPO.RTM. vectors are provided in the Invitrogen
Vectors>TOPO Vectors folder in VectorDesigner.
[0439] Selecting Vectors in the TOPO.RTM. Cloning Workspace
[0440] If the TOPO.RTM. vector is in the VectorDesigner database,
you can select it in the TOPO.RTM. Cloning workspace. Click on the
Browse in Vector button in the workspace. The window will expand,
displaying navigation tools at the bottom. Using the folder tree in
the left-hand part of the window, navigate to the folder containing
your vector. Click on the vector name in the right-hand part of the
window. The vector will be added to the Vector field in the
workspace. (Note that you may need to scroll up in the window to
view the Insert field.)
[0441] Selecting Vectors in the Molecule Viewer
[0442] From the Molecule Viewer, you can transfer the TOPO.RTM.
vector to the workspace. Go to the Cloning>TOPO Cloning menu and
select Add Molecule to Workspace as Vector. The TOPO.RTM. Cloning
workspace window will be displayed and the selected sequence will
be listed in the Vector field.
Creating the New Molecule
[0443] After you have added a compatible insert to the Insert field
and a compatible vector to the Vector field, the Clone button in
the Molecule Construction workspace will become active. If you
select inserts and/or vectors with incompatible termini, an alert
message will appear in the left-hand pane of the Molecule
Construction workspace, and you will be prompted to select
different inserts/vectors. Click on Clone to create the new
expression clone. The molecule will open in a new Molecule Viewer
window. Use the Save command in the Molecule Viewer to save the new
molecule. Information about the New Molecule Any features from the
constituent molecules will be preserved in the new molecule, except
for features that may be eliminated in the recombination reaction
(e.g., a TA overhang feature).
[0444] In addition to the standard information provided in the
Molecule Viewer, the following information is provided for
constructed molecules: In the Text Pane, the Design Description
outlines the steps for the appropriate cloning reaction. In the
Text Pane, the Component Fragments folder provides a description of
each molecule fragment used to construct the molecule. Under each
fragment, click on Open in Molecule Viewer to open the fragment in
a new Viewer window (note that the fragment in the new Viewer
window will not be saved).
Analysis of the New Molecule
[0445] You can now analyze the expression clone using the open
reading frame and sequence translation analysis tools in
VectorDesigner to verify that the DNA sequence is inserted and will
be expressed as intended.
CloneRanger.TM.
[0446] You can search Invitrogen's online clone collection for a
specific DNA target sequence using the online Web tool
CloneRanger.TM.. VectorDesigner can link to CloneRanger.TM. and
automatically enter a selected target sequence into the search
field.
[0447] To use CloneRanger.TM., in the Molecule Viewer dialog:
Select the part of the molecule sequence that you want to search
for, or make no selection if you want to search for the entire
molecule sequence. Click on the CloneRanger button (CloneRanger) on
the main toolbar, or select the command from the Tools menu. The
CloneRanger.TM. Web site will open, and a BLAST search for the
sequence will be automatically initiated. When the search is
complete, the BLAST search results page will be displayed. At this
point, you can: Use the tools in CloneRanger.TM. to select and
order the desired clone; select the desired clone and click on Send
to VectorDesigner to import the clone sequence back into
VectorDesigner. See Importing Clones for more information.
Importing Clones from CloneRanger.TM.
[0448] If you have identified one or more clones containing your
sequence of interest in Invitrogen's CloneRanger.TM. Web tool, you
can click on Send to VectorDesigner in the CloneRanger.TM. results
page to import the clone sequence(s) into VectorDesigner for
analysis.
[0449] After you click on Send to VectorDesigner in
CloneRanger.TM., the Import Clones window will open in
VectorDesigner. In the window, the Clone ID, Sequence, and
Collection for each clone will be displayed in the right-hand pane.
In the left-hand folder tree, select the folder or subfolder in
which to save the clone sequence(s). Clone sequences can be saved
as DNA molecules in the DNA/RNAs main user folder or subfolders. To
create a new folder, select the Create a New Folder checkbox and
enter the folder name in the field. Select the appropriate option
under If Object Already Exists--Rename, Overwrite, or Do Not
Import. If you select Rename, and the object name already exists in
the database, VectorDesigner will automatically rename the new
molecule with a numerical extension (1, 2, 3, etc.). When you have
made your selections, click on Import. The Import Results page will
confirm the results of the import. Click on Return to Database
Browser to go to the Database Browser window. At this point you can
navigate to the folder in which you saved the clone(s) and open
each clone in a Molecule Viewer window. Clones are imported as
linear DNA molecules.
OligoPerfect.TM. Designer
[0450] You can design primers for molecule construction and other
applications using tools within VectorDesigner (see Designing
Primers), or you can send a target DNA sequence from VectorDesigner
to the online Web tool OligoPerfect.TM. Designer to design and
order primers. OligoPerfect.TM. Designer has its own primer design
algorithms and procedures. See the OligoPerfect.TM. Web page and
online Help for detailed information and instructions.
[0451] To input a target sequence into OligoPerfect.TM., in the
Molecule Viewer dialog: Select the part of the molecule sequence
for which you want to design primers, or make no selection if you
want to design primers for the entire molecule sequence. Click on
the OligoPerfect button (OligoPerfect) on the main toolbar, or
select the command from the Tools menu. The OligoPerfect.TM. Web
site will open, and the sequence you selected will be entered in
the Target Sequence field. Your login name and the name of the
target sequence will also be automatically entered. The
OligoPerfect.TM. Designer will guide you through the primer design
process.
[0452] In the primer design results page, you can: Select and order
the desired primer designs. Select the desired primer designs and
click on Send to VectorDesigner to import the primer sequence(s)
back into VectorDesigner. See Importing Primers for more
information.
[0453] Importing Primers from OligoPerfect.TM.
[0454] If you have identified primer designs for your sequence of
interest using Invitrogen's OligoPerfect.TM. Designer, you can
click on Send to VectorDesigner in the OligoPerfect.TM. results
page to import the primer sequence into VectorDesigner for
analysis.
[0455] After you click on Send to VectorDesigner in
OligoPerfect.TM., the Import Primers window will open in
VectorDesigner. In the window, the primer name, sequence, and other
information from OligoPerfect.TM. will be displayed in the
right-hand pane. In the left-hand folder tree, select the database
folder or subfolder in which to save the primer sequence(s).
Primers can be saved in the Primers main user folder or subfolders.
To create a new folder, select the Create a New Folder checkbox and
enter the folder name in the field. Select the appropriate option
under If Object Already Exists--Rename, Overwrite, or Do Not
Import. If you select Rename, and the object name already exists in
the database, VectorDesigner will automatically rename the new
molecule with a numerical extension (1, 2, 3, etc.). When you have
made your selections, click on Import. The Import Results page will
confirm the results of the import. Click on Return to Database
Browser to go to the Database Browser window. At this point you can
navigate to the folder in which you saved the primers and open them
in the Edit Primer Properties dialog.
Performing a BLAST Search
[0456] BLAST (Basic Local Alignment Search Tool) searches compare
the similarity of a particular DNA or protein sequence to verified
gene and protein sequences in multiple public databases. For
detailed information on BLAST search types, settings, parameters,
search databases, etc., see the BLAST search information page at
NCBI.
[0457] Using VectorDesigner, you can automatically perform a BLAST
search of NCBI databases for all or part of a nucleotide or protein
molecule sequence. In the Molecule Viewer window: Select the part
of the sequence--that you want to search for, or make no selection
if you want to search for the entire molecule sequence. Click on
the BLAST Search button (blast) on the main toolbar, or select the
command from the Tools menu. The BLAST Search dialog will open. In
the dialog, under Sequence Range, select Whole Sequence to search
for the whole sequence, or Selection Only to search for a portion
of the sequence you have selected. Under Sequence Strand, select
Direct to search for the direct strand sequence, or Complementary
to search for the complementary strand sequence. Under BLAST Page,
select the type of database you want to search. See the NCBI BLAST
search page for more information on the different search types. For
protein sequences, you can search Proteins or Translations
databases. For nucleotide sequences, you can search Translations,
Nucleotides, or MegaBLAST databases. When you have made your
selections, click on OK. The search window for the selected NCBI
database will open, and the sequence will appear pasted in the
search field. Select any additional search parameters in this
window and perform the search.
Analysis Pane
[0458] The Analysis Pane displays graphical plots of a variety of a
DNA and protein sequence analyses. You can display multiple plots
at a time in the Analysis Pane. The available analyses depend on
the molecule type (DNA/RNA or protein). The Analysis Pane and the
Graphics Pane are displayed in the same pane in the Molecule
Viewer. The Graphics Pane is displayed by default. To display the
Analysis Pane, click on the Analysis Pane button below the Graphics
Pane. To return to a view of the Graphics Pane, click on the
Graphics Pane button Graph Format
[0459] The graphs in the Analysis Pane display different
physiochemical properties of the sequence. Many of properties are
based on parameters like charge that exert effects over distance.
Other properties represented in the plot depend on the way adjacent
bases/amino acids fold in 3-dimensional space, which is a function
of the sequence itself.
[0460] The vertical (Y) axis in the graph shows the values of the
analysis results; the horizontal (X) axis displays either numerical
positions in the sequence or residues. At any point along the
sequence, the Y value is derived not just from the specific residue
at that point but also from adjacent residues. Each analysis
algorithm uses an optimum window of adjacent residues to calculate
the value for a point. You can adjust this window size in the Plot
Properties dialog (see below).
Plots Setup
[0461] Use the Plots Setup dialog to select and arrange the
analysis graphs to display in the pane. To open the dialog, click
on the Plots Setup button below the Analysis Pane or select the
command from the right-click menu. In the Plots Setup dialog, the
available analyses are listed in the top window and the selected
graphs are listed in the bottom window. Analysis graphs are
displayed in panels. You can add one or more analyses to a panel,
and display multiple panels in the Analysis Pane.
[0462] To add analyses to panels: Click on an analysis name in the
Available Analyses window to select it. To select multiple
graphics, use Control+Click and Shift+Click key combinations. Click
on the Copy Analyses button next to the top window. In the bottom
window, click on a panel name in the folder tree or create a new
panel by clicking on the Create New Panel button. The panel will be
selected in the tree. Click on Paste Analyses to Panel to add the
analysis or analyses to the panel. Note that if you paste multiple
analyses to the same panel, they will be displayed in the same
graph in the Analysis Pane. To remove a panel: Click on the panel
in the bottom window. Click on Remove Panel (ELJ). All the analyses
in the panel will be removed as well.
[0463] To copy an analysis between panels: Select the analysis to
copy in the bottom window. Click on the Copy Analyses button next
to the bottom window. Select the panel you want to copy the
analysis to, and click on Paste Analyses to Panel.
[0464] To delete an analysis from a panel: Click on the analysis to
select it. Click on Remove Analysis. To reorder panels in the
Analysis Pane: Click on a panel in the bottom window. Use the arrow
buttons next to the bottom window to reorder the panels. When you
have arranged the analyses and panels in the dialog, click on OK to
display them in the Analysis Pane.
Displaying Analyses in the Analysis Pane
[0465] The Analysis Pane window includes various viewing tools: To
select a region of the sequence in both the Analysis Pane and the
Sequence Pane, drag your cursor over the sequence in either pane.
Double-click on a feature in the Text Pane to select that region of
the sequence in the Analysis Pane. To zoom in on the graphs, click
on the Zoom In button. To zoom out, click on the Zoom Out button.
To magnify a region of the graphs, drag your cursor to select the
region, then click on the Zoom Selection to Window button. To fit
the graphs lengthwise to the current window, click on Fit to Window
button. To fit the graphs vertically to the current window,
right-click in the pane and select Fit to Size. To make the panels
all the same size within the window, right-click in the pane and
select Distribute Panels. To hide or show the axes in the graphs,
click on the Hide/Show Axes button. To change the display of each
plot in the Analysis Pane, see Plot Properties, below.
Plot Properties
[0466] The Plot Properties dialog controls how each plot is
displayed in the graph. To open the dialog, right-click on an graph
in the Analysis Pane and select Plot Properties. The dialog is
divided into three tabs. When you have made your selections, click
on OK.
[0467] Diagram Tab: Click on the Graph Color button to open a
dialog in which you can select a plot color and/or adjust the
Red-Green-Blue (RGB) values of the color. Select the Draw Type from
the dropdown list. Min-Max-Average displays the calculated minimum,
maximum, and average values over each analysis region within the
sequence as levels of shading along the line of the graph. Under
Preprocess Type, select Linear Interpolation to provide a linear
interpolation of the graph line, or No Preprocessing to display the
line without interpolation.
[0468] Params Tab: Window Size is the size of the processing
"window" used to scan the sequence for analysis. Enter a number of
bases/amino acids in the Window Size field (see example below).
Step Size is the number of bases/amino acids in a sequence that
constitute an analysis point in the plot. Enter number of
bases/amino acids in the Step Size field. For example, if you
select a % GC Content analysis with a window size of 21 and a step
size of 1, the GC content percentage will be calculated for a
21-base region centered on each base in the sequence (10 bases on
either side of the base). A step size of 5 would calculate the
percentage for a 21-base region centered on each 5-base region in
the sequence.
[0469] The Info tab provides information on the type of analysis in
the plot, including any references to external literature.
Links to Resources and Ordering: Links to Additional Resources
[0470] VectorDesigher includes built-in links to Web tools, Web
sites, download pages, and product ordering pages.
Links to Web Tools and Software Downloads
[0471] From the Software>Desktop Products menu, select:
Information on Desktop Software to link to a Web page with
information on Invitrogen's suite of bioinformatics software,
including VectorNTI Advance.TM. for molecule construction,
analysis, and databasing; Vector Xpression.TM. for microarray
analysis and databasing; and Vector PathBlazer.TM. for biological
pathways analysis. Download VectorNTI Advance for PC to link to a
download page for VectorNTI Advance.TM. for the Microsoft
Windows.RTM. operating system. Download VectorNTI Suite for Mac OS
X to link to a download page for VectorNTI Suite.TM. for the
Macintosh.RTM. OS X operating system. Download Vector Xpression 3.0
to link to a download page for Vector Xpression.TM. 3.0 software
for Microsoft Windows.RTM.. Download Vector PathBlazer to link to a
download page for Vector PathBlazer.TM. software for Microsoft
Windows.RTM..
[0472] From the Software>Web Tools menu, select: RNAi Designer
to design custom RNAi molecules, including Stealth.TM. RNAi oligos,
for gene knockdown experiments; Peptide Designer to design custom
peptides from a protein target sequence; LUX Designer to design
custom LUX.TM. Primer sets from a DNA target sequence for real-time
quantitative PCR and RT-PCR applications. Additional Web tools are
listed under the Tools menu and include the following: BLAST
Search; OligoPerfect Designer; CloneRanger.
[0473] Links to Molecule Information
[0474] Certain types of imported molecules and example molecules
from Invitrogen include links to additional information: Text Pane:
The Links folder in the Text Page of the Molecule Viewer provides a
list of links to additional online resources for the molecule. The
Feature Map folder may also contain Links folders in the individual
Feature folders with links to information about each feature. The
Imported Features Not Shown on Map folder may also contains Links
folders for individual features. Double-click on a link to open it.
Feature List: Right-click on a feature in the list and select Open
Link to access a list of links to online databases with information
about the feature. Select a link from the list to open it. A link
can launch a new browser window or an email application. Note that
you cannot create new links using VectorDesigner.
[0475] Links to Invitrogen Products: You can order primers,
vectors, restriction enzymes, and related products from Invitrogen
using links in VectorDesigner.
[0476] For example, the user can order primer designs from the
Molecule Viewer, or you can order saved primers from the Database
Browser. If you have primer designs in the Molecule Viewer, go to
the PCR Primers folder in the Text Pane, open the Product folder
containing the designs, and click on the Order from Invitrogen link
next to each primer name. You will be prompted to use the existing
primer name or enter a new one (this will not change the primer
name in the Molecule Viewer), and the primer sequence will
automatically be loaded into Invitrogen's ordering system. You can
specify the details of your order (purity, synthesis scale, etc.)
on the Web site.
[0477] If you have saved primers in the VectorDesigner database, go
to the Primers folder in the Database Browser, select the checkbox
next to each primer that you want to order, and click on the Order
button. Each primer sequence will automatically be loaded into
Invitrogen's online ordering system. You can specify the details of
each primer order (purity, synthesis scale, etc.) on the Web
site.
Vectors
[0478] You can order Invitrogen vectors and related products from
VectorDesigner. VectorDesigner also provides ordering links for
molecules constructed from Invitrogen vectors. In the Database
Browser, an Add to Cart button will be available in the Order
column for each Invitrogen vector or vector constructed from an
Invitrogen vector. Click on the button to open an Invitrogen
catalog page with information about products related to the vector.
In the Molecule Viewer, an Invitrogen Products link will be
available in the Text Pane for each Invitrogen vector or vector
constructed from an Invitrogen vector. Click on the link to open an
Invitrogen catalog page with information about products related to
the vector.
[0479] Restriction Enzymes Restriction enzymes sold by Invitrogen
will be flagged by a symbol in the Restriction Map folder of the
Text Pane. Click on the Order from Invitrogen link next to the
enzyme name to open an Invitrogen catalog page with information
about that enzyme.
Registration
[0480] The user may be propted to fill out the information in the
Registration form and create a User Name and Password to use
VectorDesigner. The User Name and Password will give you secure
access to all the molecules in the VectorDesigner database. The
molecules in your private user folders will only be accessible
using your User Name and Password.
Browser and Operating System Requirements
[0481] VectorDesigner is supported on various operating systems,
Internet browsers, and Java systems: Java Applet and Security
Warning
[0482] VectorDesigner uses a Java applet to display viewers and
dialog boxes. In order for the Java applet to run, it may require
access to files and other resources on your computer. Depending on
the permissions settings for your computer or your network system,
you may receive a Security Warning when the Java applet
initializes.
Security
[0483] All molecule sequences, user information, and other data are
encrypted during transmission and transmitted via a secure socket
layer (SSL). They are stored in encrypted form on our secure
servers behind multi-tiered firewalls. Sequences in the private
user folders are accessible only if you log in with the correct
user name and password.
[0484] Privacy: For detailed information about Invitrogen's privacy
policy, click on the Privacy Policy link at the bottom of any page
in the VectorDesigner.
Dialog Boxes and Notes Add/Edit Feature
[0485] Use this dialog to define the various features in a
molecule, including promoter regions, open reading frames, binding
sites, epitopes, or any other region of interest. In the dialog,
the Feature Type field lists the available feature types in the
database for the molecule. Select a feature type from the list. If
you cannot find the precise type you are looking, select Misc.
Feature. Note that you cannot add new feature types in
VectorDesigner. Enter a name for the feature in the Feature Name
field. Select the format to use for defining the sequence region:
Use Start.End Format or Use Start . . . Length Format. If you
selected or marked the feature region in the sequence before
opening the dialog, the start and length/endpoint of the feature
will be automatically entered in the dialog. To change the region,
enter the start and length/endpoint in the fields. For features
with multiple components (i.e., internal start and endpoints),
select Multi-component and enter each start and length/endpoint in
the field. Use the following format: <start1> . . .
<length/endpoint1>, <start2 . . . length/endpoint2>,
etc.
[0486] Click on Reset to Selection to undo any changes you may have
made to a preselected sequence region. Click on Reset to Mark to
undo any changes you may have made to a marked sequence region.
Select the Complementary checkbox if the feature is located on the
complementary molecule strand. Note: VectorDesigner uses the
currently accepted convention for calculating the coordinates of
complementary features. All coordinates are given as if on the
direct strand, from left to right in the sequence. Enter a
description for the feature in the Description field. When you have
made your selections, click OK to add the feature.
Annotate Analysis dialog
[0487] Use this dialog to define an open reading frame, restriction
fragment, or primer as a feature. In the dialog: Select the feature
type from the Feature Type dropdown list; enter the feature name in
the Feature Name field; enter a description in the Description
field; click on OK. The feature will be added to the feature map.
For primers and ORFs, if you want to alter the start and/or
endpoint of the sequence before defining it as a feature,
right-click on the primer or ORF and select Annotate Analysis Item.
This will open the Add/Edit Feature dialog, in which you can change
the start/endpoint of the feature.
[0488] BLAST Search: Use this dialog to perform a BLAST search of
NCBI databases for all or part of a nucleotide or protein molecule
sequence. In the dialog: Under Sequence Range, select Whole
Sequence to search for the whole sequence, or Selection Only to
search for a portion of the sequence you have selected. Under
Sequence Strand, select Direct to search for the direct strand
sequence, or Complementary to search for the complementary strand
sequence. Under BLAST Page, select the type of database you want to
search. See the NCBI BLAST search page for more information on the
different search types. When you have made your selections, click
on OK. The search window for the selected NCBI database will open,
and the sequence will appear pasted in the search field. Select any
additional search parameters in this window and perform the
search.
Browse to Primer Folder
[0489] Use this dialog to locate the database folder containing the
desired primer sequences. Highlight the folder in the directory
tree and click on OK to select the folder. Choose
Direct/Complementary Strand Addition
[0490] Use this dialog box to add any additional nucleotides or
specific sequences to the 5' end of the direct or complementary
primer. Access this dialog by clicking on the Browse button next to
the Direct and/or Complementary fields in the PCR Analysis dialog.
In the dialog, you can select from any or all of the following
options: Type the nucleotides you want to add directly into the
field. Double-click on one or more defined sequences in the table
below the field. If you double-click on more than one defined
sequence, the defined sequences will be added to the field above 5'
to 3' in the order in which you select them. You can then edit the
complete sequence in the field. To add a restriction endonuclease
cut site at the 5' end of the sequence addition, select the Add One
REN Site 5' to the Additions Above checkbox, and select the
restriction enzyme from the list below. Depending on the length of
the cut site sequence, a pop-up box may prompt you to add
nucleotides to the site to improve efficiency of the REN cleavage.
Note that you can only add a single restriction site to the 5' end
of the primer using this method. When you have made your
selections, click on OK. The sequence additions will be displayed
in the PCR Analysis dialog.
[0491] Create New Folder: Use this dialog to create new subfolders
within the three main user folders in the database. Enter the new
folder name in the Name field and a folder description in the
Description field. Click on Save to create the folder.
[0492] Molecule: Use this dialog to create a new molecule based on
the molecule currently displayed in the Molecule Viewer. You can
create a new molecule from a selected area of the existing
molecule, such as a restriction fragment, or from the whole
molecule. From DNA or RNA molecules, you can create DNA/RNA
molecules that are the reverse complement of the existing molecule
or you can create protein molecules from a translation of the
sequence.
[0493] In the dialog: Enter a name for the new molecule in the Name
field, and a description (if any) in the Description field. Next,
specify which part of the existing molecule to use as the basis for
the new molecule. If you selected or marked a region of the
existing molecule before you opened the dialog, the Selection or
Mark options will be available and selected. Otherwise, select
Molecule to select the whole molecule or Specified Range to enter
the sequence range in the From and To fields. DNA/RNA molecules
only: Select the Reverse Complement checkbox to create a molecule
from the complementary sequence. Select Translate to create a
protein molecule from a translation of the sequence. When you have
made your selections, click on OK. The new molecule will be created
and displayed in a new Molecule Viewer window. The new molecule
will not be saved. To add the molecule to the database, you must
save it.
Edit Primer Properties
[0494] The Edit Primer Properties window displays the sequence,
name, and description of each primer that has been saved as a
separate molecule in the VectorDesigner database. Note that primer
designs generated using the tools in the Molecule Viewer are saved
with the DNA molecule file (see Primer Designs and PCR Products for
more information). Primers saved as separate primer files are
stored in the Primers folder in the VectorDesigner database. To
open a primer file, click on the primer name in the Primers folder
in the Database Browser. The Edit Primer Properties window includes
Name, Description, and Sequence fields. You can edit the text in
any of these fields.
[0495] To order the primer sequence from Invitrogen, click on the
Order button in the window. The primer sequence will automatically
be loaded into Invitrogen's online ordering system, where-you can
specify the details of your order (purity, synthesis scale, etc.).
To save any changes you make to the name, description, or sequence,
select Rename or Overwrite to specify whether you want to rename
the saved file or overwrite the existing file. Then click on the
Save button. If you select Rename, the primer will automatically be
saved with the existing name plus a numerical extension (1, 2, 3,
etc.).
Enzymes List Dialog
[0496] The Enzymes List dialog enables you to create a custom list
of restriction enzymes to use in restriction mapping. In the
dialog, the Customized List lists enzymes that have been selected
for use, while the All Enzymes list shows the remaining unselected
enzymes in the database. The enzymes are listed alphabetically.
[0497] To add or remove enzymes from the Customized List. Click on
an enzyme in one of the lists to select it. Use Shift-Click and
Control-Click key commands to select multiple enzymes in the list.
Click on Add to move the selected enzymes from the All Enzymes list
to the Customized List. Click on Remove to remove the selected
enzymes from the Customized List. Alternatively, click on Add All
to move all the enzymes to the Customized List, or Remove All to
remove them from the list. Click on OK to accept your changes.
Export to File Dialog
[0498] Use the Export to File dialog to export the data for the
molecule to a file (text format) or to a separate browser window
(HTML format): In the dialog, select either Show in Browser or Save
Single Object to File. Select the export format (GenBank, FASTA,
etc.) and click on OK. If you selected Save Single Object as File,
you will be prompted to save the file or open it in a application
window. The data will be exported as an ASCII text file. If you
selected Show in Browser, the exported file will be displayed in
HTML format a separate browser window.
Export to GIF Dialog
[0499] Use the Export to GIF dialog to export the molecule image as
it is displayed in the Molecule Viewer as a GIF image. Note: This
command will export only the current view of the molecule. If the
displayed information (sequence, graphics, text, etc.) is cut off
at the margins of the panes in the Molecule Viewer, the data will
appear cut off in the resulting image. Be sure to configure your
Molecule Viewer panes as desired--for the resulting image. With
your molecule displayed in the Viewer, go to the Molecule menu and
select Export to GIF. In the Export to GIF dialog, select Whole
Viewer to export an image of the entire Molecule Viewer window, or
select the specific pane that you want to export. Select Draw
Border to include a border line around the image. If you are
exporting the Graphics Pane only, select Graphics Only if you do
not want to include the toolbar at the bottom of the pane. When you
click on OK, you will be prompted to save the GIF file or open it
in an application window.
Map is Updated
[0500] If you make changes to a molecule sequence in the Molecule
Viewer, and those changes affect defined features in the molecule,
the Feature Map is Updated dialog will open. In this dialog you can
remove any or all of the defined features that will be changed.
Note that this will not alter the change that you are making to the
sequence; it will only remove the defined feature(s) affected by
the change.
[0501] In the dialog, the affected features are listed. Select a
feature in the list and click on Delete to flag it for deletion. To
delete all the features in the list, click on Delete All. If you
change your mind, select the feature flagged for deletion and click
on Keep, or click on Keep All to keep all features. Click on OK to
make the sequence change. If you flagged a feature for deletion in
the dialog, that feature will be removed.
[0502] Find Sequence: Use this dialog to find a sequence within a
larger sequence. In the dialog, type or paste the sequence you want
to find, specify the search direction (Up or Down), and click on
Find Next. Click on Find Next again to find the next occurrence of
the sequence within the larger sequence. Click on Close to close
the dialog.
Frequently Used Enzymes
[0503] AccI, AM, Apal, Aval, BamHI, Bglll, Clal, Ddel, Dpnl, Dral,
EcoRI, EcoRV, Haelll, Hhal, Hindi, Hindilll, Hinfl, Hpal, Hpall,
Kpnl, Mbol, Mlul, MscI, Msel, Ncol, Ndel, Nhel, NotI, Nrul, Nsil,
PinAI, PstI, Pvul, PvuII, Rsal, Sail, Seal, Smal, Spel, SphI, Sspl,
SstI, Sstll, Stul, TaqI, Xbal, Xhol Gateway.RTM. Cloning PCR
Products
[0504] In the PCR Analysis: Gateway Cloning dialog, VectorDesigner
will add attB extensions to the direct and complementary primers to
generate the af/B-PCR product required for BP recombination into a
Gateway.RTM. entry clone. Note that which extensions are added to
the direct and complementary primers will depend on your Cloning
Strand selection. Consult the Gateway.RTM. Technology manual for
more information about designing primers for Gateway.RTM.
cloning.
[0505] Gateway.RTM. cloning will automatically add a 5' sequence to
the forward primer consisting of four guanine (G) residues at the
5' end followed by a 25-bp attB1 site. It will also add a 5'
sequence to the reverse primer consisting of four G residues at the
5' end followed by a 25-bp attB2 site. See Important Note About
Reading Frames for details on preserving the reading frame in
af/B-PCR products. TABLE-US-00001 (SEQ ID NO:10) attB1 Forward
primer: 5'-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-T-
(template-specific sequence)-3' (SEQ ID NO:11) atiB1 Reverse
primer: 5'-GGGG-AC-CAC-TTT-GTA-CAA-GAA-AGC-TGG-GT-
(template-specific sequence)-3'
Note About Reading Frames
[0506] For cloning applications, if you want to fuse your PCR
product in frame with an N- or C-terminal peptide tag in the
vector, you may need to add bases to the PCR primers to maintain a
continuous reading frame between the tag and the insert. To add
bases to the primers, use the Choose Direct/Complementary Strand
Addition dialog box.
[0507] Gateway Cloning Examples: In Gateway.RTM. cloning, to fuse
your a<<B-PCR product in frame with an N-terminal tag, you
must add 2 bases immediately after the attB1 addition (i.e., at the
3' end of the addition). These two nucleotides cannot be AA, AG, or
GA, because these additions will create a translation termination
codon. To fuse your attB-PCR product in frame with an C-terminal
tag, you must add 1 base immediately after the attB2 addition
(i.e., at the 3' end of the addition), and you must eliminate any
stop codons between the a/B2 site and your gene of interest. If you
do not want to fuse the PCR product in frame with a C-terminal tag,
your gene of interest or the primer must contain a stop codon. To
add a stop codon to the primer, use the Choose Direct/Complementary
Strand Addition dialog box.
Insert Sequence
[0508] Use this dialog to insert a new sequence into an existing
sequence in the Molecule Viewer. First, be careful to click at the
point in the existing sequence where you want to insert the new
sequence. In the dialog, note the insertion point listed below the
field. Type or paste the new sequence into the dialog and click on
OK. Note: Use only standard code letters when entering the
sequence. Nonstandard characters will be marked with a ? in the
Insert Sequence dialog and you will be prompted to remove them
before adding the new sequence. If you are adding the sequence
within a defined feature, the Feature Map is Updated dialog will
open, listing the features in the molecule that will be affected by
the insertion. In this dialog you can remove any or all of the
defined features that will be changed. Note that this will not
alter the change that you are making to the sequence; it will only
remove the defined feature(s) affected by the change. Click on OK
to make the changes.
[0509] MegaBLAST uses a "gTeedy algorithm" (Webb Miller et al., J
Comput Biol February-April; 2000 7(1-2):203-14) for nucleotide
sequence alignment searches and concatenates many queries to save
time scanning the database. It is optimized for aligning sequences
that differ slightly and is up to 10 times faster than more common
sequence similarity programs. It can be used to quickly compare two
large sets of sequences against each other. MegaBLAST permits
searching with batches of ESTs or with large cDNA or genomic
sequences.
Molecule Construction PCR Products
[0510] In the PCR Analysis: Molecule Construction dialog, under
Cloning Termini, if you select: Blunt: No extensions or overhangs
will be automatically added; TA: 3' A extensions will be
automatically added to both ends of the PCR product, for TA cloning
into an appropriate linearized expression vector with T overhangs.
Note that no extensions will be added to the primers. Rather,
VectorDesigner will account for the nontemplate-dependent terminal
transferase activity of Taq DNA polymerase that adds a single
deoxyadenosine (A) to the ends of the PCR products.
ORF Search
[0511] Use this dialog to identify open reading frames (ORFs) in a
DNA molecule. Using the tool, you set the minimum ORF size, the
start and stop codons to search for, and other parameters, and
VectorDesigner will generate a list of defined ORFs and highlight
them in the sequence. In the ORF Search dialog: Specify the Minimum
ORF Size (in codons) and select the Nested ORFs checkbox if you
want to search for nested ORFS (ORFs that have the same stop codon
but different start codons). In Start Codons and Stop Codons
fields, enter one or more start and stop codons to search for when
identifying ORFs. Separate each codon by a space. To reset the
fields, click on Reset to Default. Select Include Stop Codon in ORF
if you want the stop codon to be considered part of the ORF.
Otherwise, the stop codon will not be included in each ORF defined
in the sequence. Click on OK to search for the ORFs. The ORFs will
be marked on the sequence in the Graphics Pane and a folder called
Open Reading Frames will be created in the Text Pane.
[0512] PCR Analysis: Use this dialog to design PCR primers from a
target sequence for cloning applications (including TOPO.RTM.
Cloning and Gateway.RTM. Cloning) or PCR analysis of a DNA molecule
fragment.
[0513] In the dialog, the default values and available options will
different slightly depending on the application you selected (these
differences are noted below). Under the Primer Definition and
Construction tab, the From and To fields define the region that
will be analyzed for primer designs. You can change the numbers in
these fields.
[0514] Next, enter the primer design parameters, or select the
folders containing the saved primers that you want to evaluate for
compatibility with the molecule sequence. The following fields are
only available if you selected Design Primers to Amplify Selection
when you opened the dialog: To include primer design regions before
and after the target sequence, enter a number of bases in the
Before and After fields. Maximum # of Outputs: Enter the maximum
number of primer pair designs to generate. Note that VectorDesigner
may generate fewer designs if no more can be found. Tm: Enter the
limits in degrees Celsius for primer melting temperature (Tm)
(temperature at which 50% of primer is a duplex) in the Minimum and
Maximum fields. Designs with Tin's outside this range will be
excluded. % GC: Enter the maximum and minimum percent GC content
for the primers in the fields. Designs with a percent GC content
outside this range will be excluded. Length: Enter the maximum and
minimum length (in bases) of each primer in the fields. Designs
that fall outside this range will be excluded. Nucleotide sequences
such as RENs attached to a primer's 5' end are included when
calculating primer length. Exclude Primers with Ambiguous
Nucleotides: If your sequence includes ambiguous bases (i.e., code
letters other than A, G, C, T), select this checkbox to exclude
regions containing these bases from the primer design search.
[0515] The following fields are only available if you selected Find
Amplicon in Sequence Using Existing Primers when you opened the
dialog: Click on the Direct button to select the folder containing
the direct primers that you want to evaluate, and click on
Complementary to select the complementary primers to evaluate. The
Browse to Primer Folder dialog will open when you click on each
button. Select the folder and click on OK. Enter a percentage
similarity in the Similarity>=Threshold field. Each primer
sequence must be at least this similar to the molecule sequence to
be selected by the designer. Select the checkbox next to
LastNucleotides Must Have 100% Similarity to specify a number of
nucleotides at the 3' end of each primer that must be 100% similar
to the target sequence. Enter a number of nucleotides in the
field.
[0516] Next, select the conditions of the PCR reaction you are
performing. If you are unsure of these values, use the default
values: Salt cone: The salt concentration of the PCR reaction, in
mMol. If you are unsure, use the default value of 50.0. Probe cone:
The final concentration of each primer in the reaction, in pMol. If
you are unsure, use the default value of 250.0. dG temp: The
temperature of the free energy value of the reaction, in degrees
Celsius. If you are unsure, use the default value of 25.0.
[0517] Under Cloning Termi, select the type of PCR product you are
generating. The available options will vary depending on your
cloning application. Click on an application below for more
information on how the primer and/or PCR product will be modified
based on your selection: e.g., TOPO.RTM. Cloning PCR Products;
Gateway.RTM. Cloning PCR Products; Molecule Construction PCR
Products.
[0518] For cloning applications, under Cloning strand, select the
strand whose sequence will be expressed: Direct or Complementary.
Note that this will affect the primer strand to which Directional
TOPO.RTM., Gateway.RTM., and other primer additions are added.
[0519] Next, select additions to each primer. Click on the Browse
button next to the Direct and/or Complementary fields. The Choose
Direct/Complementary Strand Addition dialog will open. Select the
strand additions in the dialog and click on OK. The additions will
be listed in the appropriate field. Additions to the primer
sequence will not be used in calculations of primer Tm, % GC, etc.
If you change the Cloning Strand (step above) after selecting the
primer additions, the additions will switch to the other
strand.
[0520] Click on the Pairing, Structure and Uniqueness tab to access
additional primer specifications. Max. Tm Difference: Specify the
maximum difference in melting temperature between sense and
antisense primers in degrees Celsius. Note the differences in GC
content between the two primer regions of the sequence when
specifying this difference; a difference that is too small may
result in no primers being found. Max. % GC Difference: Specify the
maximum percentage difference in GC content between sense and
antisense primers. Note the differences in GC content between the
two primer regions of the sequence when specifying this difference;
a difference that is too small may result in no primers being
found. Primer-Primer Complementarity: Permitted with dG>=:
Select this checkbox and enter the minimum permitted value for free
energy of a primer-primer duplex. Primer pairs which have a free
energy value>/=to this number will be accepted. Primer-Primer
Complementarity: 3' End Permitted with dG>=______: Select this
checkbox and enter the minimum permitted value for free energy of
complementarity between the 3'-end of the primers (the final 5
bases of each primer will be evaluated). Primer pairs which have a
3'-end complementarity free energy value>/=to this number will
be accepted. Exclude Primers With: In the Repeat field, enter the
maximum number of base-pair repeats allowed in each primer. In the
Palindrome field, enter the maximum permitted length of palindromes
in each primer sequence. In the Hairpin Loops field, enter the
minimum permitted value for free energy of hairpin loops within
each primer. Primer Uniqueness: Select this checkbox to reject
primers above a certain percentage similarity to secondary sites
within either the entire sequence or within the amplicon. Enter an
percentage similarity in the field, and select Within Entire
Sequence or Within Amplicon Only.
[0521] Click on OK to design the primers. You will be prompted to
send the PCR product for the first (highest ranked) primer pair
directly to the appropriate molecule construction workspace as an
insert. If you click on No, all the primer pairs generated will be
added to the PCR Primers folder in the Text Pane of the Molecule
Viewer. Plot Properties
[0522] The Plot Properties dialog controls how each plot is
displayed in the Analysis Pane. The dialog is divided into three
tabs. When you have made your selections, click on OK. Diagram Tab.
Click on the Graph Color button (mm) to open a dialog in which you
can select a plot color and/or adjust the Red-Green-Blue (RGB)
values of the color. Select the Draw Type from the dropdown list.
Min-Max-Average displays the calculated minimum, maximum, and
average values over each analysis region within the sequence as
levels of shading along the line of the graph. Under Preprocess
Type, select Linear Interpolation to provide a linear interpolation
of the graph line, or No Preprocessing to display the line without
interpolation.
Params Tab
[0523] Window Size is the size of the processing "window" used to
scan the sequence for analysis. Enter a number of bases/amino acids
in the Window Size field (see example below). Step Size is the
number of bases/amino acids in a sequence that constitute an
analysis point in the plot. Enter number of bases/amino acids in
the Step Size field (see example below).
[0524] For example, if you select a % GC Content analysis with a
window size of 21 and a step size of 1, the GC content percentage
will be calculated for a 21-base region centered on each base in
the sequence (10 bases on either side of the base). A step size of
5 would calculate the percentage for a 21-base region centered on
each 5-base region in the sequence.
[0525] The Info tab provides information on the type of analysis in
the plot, including any references to external literature.
[0526] Plots Setup: Use the Plots Setup dialog to select and
arrange the analysis graphs to display in the Analysis Pane. In the
Plots Setup dialog, the available analyses are listed in the top
window and the selected graphs are listed in the bottom window.
Analysis graphs are displayed in panels. You can add one or more
analyses to a panel, and display multiple panels in the Analysis
Pane.
[0527] To add analyses to panels: Click on an analysis name in the
Available Analyses window to select it. To select multiple
graphics, use Control+Click and Shift+Click key combinations. Click
on the Copy Analyses button next to the top window. In the bottom
window, click on a panel name in the folder tree or create a new
panel by clicking on the Create New Panel button. The panel will be
selected in the tree. Click on Paste Analyses to Panel to add the
analysis or analyses to the panel. Note that if you paste multiple
analyses to the same panel, they will be displayed in the same
graph in the Analysis Pane.
[0528] To remove a panel: Click on the panel in the bottom window.
Click on Remove Panel. All the analyses in the panel will be
removed as well.
[0529] To copy an analysis between panels: Select the analysis to
copy in the bottom window. Click on the Copy Analyses button next
to the bottom window. Select the panel you want to copy the
analysis to, and click on Paste Analyses to Panel B
[0530] To delete an analysis from a panel: Click on the analysis to
select it. Click on Remove Analysis.
[0531] To reorder panels in the Analysis Pane: Click on a panel in
the bottom window. Use the arrow buttons next to the bottom window
to reorder the panels. When you have arranged the analyses and
panels in the dialog, click on OK to display them in the Analysis
Pane.
[0532] Restriction Map Search: Use this dialog to identify the
restriction enzyme cut sites in a DNA molecule using a built-in
database of restriction enzymes. In the dialog: Select the category
of enzymes that you want to use from the Use Enzymes list:
Frequently Used Enzymes have been identified by Invitrogen. Click
here for a list. 7+Cutters, 6 Cutters, 5 Cutters, etc. refer to the
number of base pairs in the recognition site of each enzyme.
Enzymes in the 5' Overhang category result in fragments with a 5'
overhang; enzymes in the 3' Overhang category result in fragments
with a 3' overhang. If you select Customized, click on the
Customize button to select the particular enzymes you want to use.
The Enzymes List dialog will open. Next, enter a number in the
Display Enzymes with <=Recognition Sites field. The Designer
will analyze the sequence and use only those enzymes with less than
or equal to that number of cut sites. Alternatively, select
Unlimited to not filter the enzyme list by number of cut sites. 4.
When you have made your selections, click on OK.
Save As
[0533] In the Save As dialog: Click in the folder tree to select
the folder or subfolder where you want to save the molecule. Note
that the molecule type determines which main user folder you can
save it in (e.g., DNA/RNA molecules can only be saved in the
DNA/RNAs folder or subfolders; primers can only be saved in the
Primers folder or subfolders). To create a new subfolder within the
main folder, click on Create New Folder and enter the information
in the Create New Folder dialog. Enter a name for the molecule and
click on OK to save it to the database. The new molecule will be
listed in the Database Browser.
Sequence Properties
[0534] Use this dialog to change how the sequence is displayed in
the pane. The dialog contains following display options:
Sequence Representation Styles
[0535] Multiline Fixed: Display a fixed number bases/amino acids
per line on multiple lines, regardless of window size. (Dependent
on Symbols in Group and Groups in Line settings.). Multiline
Variable: Display a variable number of bases/amino acids per line
on multiple lines, depending on window size. Single Line: Display a
single line of bases/amino acids, regardless of window size. Show
Direct Strand Only: DNA molecules only--Select this checkbox to
show only the direct DNA strand in the pane. Symbols in Group:
Enter the number of bases/amino acids to display in a group for
ease of reading; dependent on Insert Gaps Between Groups to view
the groups in the display. Groups in Line: Enter the number of
groups to display on a line if the Multiline Fixed setting is
selected. Insert Gaps Between Groups: Select this checkbox to
insert a space between groups in the sequence.
Feature Representation Style
[0536] Show Direct Features: For protein molecules, select this
checkbox to mark defined features in the sequence with colored bars
above the sequence. For DNA molecules, this marks defined features
on the direct strand with colored bars above the sequence. Show
Complementary Features: For DNA molecules only, select this
checkbox to mark defined features on the complementary strand with
colored bars below the sequence. Feature Height: Enter a relative
height scale (1-5) for feature bars as displayed in the Sequence
Pane.
Selection
[0537] Use this dialog to select part of the sequence defined by
the start and end bases/amino acids. Enter the number of the
starting base/amino acid in the Start field and the ending
base/amino acid in the End field and click on OK. The defined area
will appear selected in the Graphics and Sequence Panes.
TOPO.RTM. Cloning PCR Products
[0538] In the PCR Analysis: TOPO Cloning dialog, under Cloning
Termini, if you Blunt: No extensions or overhangs will be
automatically added. PCR products generated using these primers are
suitable for Zero Blunt.RTM. TOPO.RTM. PCR Cloning; TA: 3' A
overhangs will be added to both ends of the resulting PCR product.
These PCR products are suitable for TOPO.RTM. TA Cloning. Note that
no extensions will be added to the primers themselves. Rather,
VectorDesigner will account for the nontemplate-dependent terminal
transferase activity of Taq DNA polymerase that adds a single
deoxyadenosine (A) to the ends of the PCR products. If the user
selects Directional, a CACC sequence will be added to the 5' end of
the direct or complementary strand primer, depending on your
Cloning Strand selection. PCR products generated using these
primers are suitable for Directional TOPO.RTM. Cloning.
[0539] Types Filter: Use this dialog to filter the types of
features highlighted in the Sequence Pane. In the dialog, deselect
the checkboxes next to the filters that you do not want to view in
the Sequence Pane, and click on OK to make the changes.
[0540] Various embodiments of the present invention have been
described above. It should be understood that these embodiments
have been presented by way of example only, and not limitation. It
will be understood by those skilled in the relevant art that
various changes in form and detail of the embodiments described
above may be made without departing from the spirit and scope of
the present invention as defined in the claims. Thus, the breadth
and scope of the present invention should not be limited by any of
the above-described exemplary embodiments, but should be defined
only in accordance with the following claims and their equivalents.
Sequence CWU 1
1
14124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1acaactttgt ataataaagt tgct 24226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2acaactttgt atagaaaagt tgggtg 26317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3ggggacaagt ttgtaca 17418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4taacatttcc aaggccat
18518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5taaccaatgg ctatgggc 186819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
6nacnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna gnnnnnnnnn nnnnttcaca
60ttctncgcng atggttgaga tgtgtataag agacagttag gtgacactat agaatacaag
120cttgcttgtt ctttttgcag aagctcagaa taaacgctca actttggcag
atccgcggcc 180gcagatctga attccggaga tttgtccnng cagatgctgc
tggccttctg ggaatcctgg 240actgtgatta ctgcgctgga gagctgttat
ctgtaactgg aagactctcc attaacctgc 300attaacaata ttgacctgga
tttcacagca gtcctataaa aagttgacta gtcacaatga 360atgtgacgag
cttgttctcc ttcaccagcc cagcagtgaa gaggctgctt ggttggaaac
420anggagacga agaagagaaa tgggcagaga aagcagtaga tgccttggtg
aaaaagctga 480agaagaaaaa aggagccatg gaggaactgg aaaaggccct
gagantgtcc tggacagccc 540agtaactgtg tcaccattcc tcgttccttg
gatggcaggc tgcaagtgtc acaccgcaag 600ggcctaccac atgtgattta
ttgccgtgtg tggcgttggc cggatctaca aagtcaccat 660gaactgaaac
ccttggagtg ctgcgagtat ccctttggtt ctaaacagaa ggaggtctgc
720atcaacccgt atcattacaa acgagtggag agtcctgtct tgccacctgt
ccttgttnca 780cggtacagtg agtacaaccc acagcacagt ctccttgcg
819747DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7gtggcgggga tcctctagag tcgacctgca
ggcatgcaag cttcagg 47868DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 8ctgtgctgtg
ggttgtactc actgtaccgt gnaacaagga caggtggcaa gacaggactc 60tccactcg
68914DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9atgtgtactc ctta 141029DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10ggggacaagt ttgtacaaaa aagcaggct 291129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11ggggaccact ttgtacaaga aagctgggt 291227DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12actgactaat ataatataca tcatcta 271369DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13ctgtgctgtg ggttgtactc actgtgcccg tggaacaagg
acangtggca agacaggact 60ctccactcg 691470DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14ctgtgctgtg ggttgtactc actgtacccg tggaacaagg
acaggtggca agacaggact 60ctccactcgt 70
* * * * *
References