The purpose of WebDSV (Web-based DNA Sequence Editor and Viewer) is to provide all the functions needed to edit and store DNA sequences, mark sequence features, carry out basic sequence analysis (restriction sites, GC-content), and graphical visualization both as marked up sequence and a map of features and restriction sites. WebDSV is designed in a way that all control elements are present in the application panels as buttons or icons rather than menus and sub-menus. The purpose of this design is to make the use of the program easy, straightforward, and intuitive. Another difference between WebDSV and a typical "offline" (installation requiring) DNA sequence editor is the separation of editor and visualization (marked up sequence) fields. This arrangement may appear a bit confusing at first glance, but it is very easy to get used to it. As double-clicking any sequence position, restriction site, amino acid symbol, or feature label in the Visualization Field will select the corresponding sequence in the Editor Field, editing a sequence and observing the result back in the Visualization Field is quite comfortable.
The WebDSV user interface consists of several parts each with data fields and/or control elements (buttons, icons). The basic layout of data fields and control elements in WebDSV is depicted in Figure 1.
Figure 1. The layout of data fields and control elements in WebDSV.
Editor Field is where the DNA sequence can be edited by typing, inserting, or deleting nucleotide characters. If you start with a sequence in the clipboard, this the place where you need to paste the sequence. After any change has been made in the sequence, click the Apply & Process button to apply the change and update the Visualization Field. If a multi-step editing that would affect a defined sequence feature needs to be done, click the Apply & Process button after each step.
Visualization Field displays the DNA sequence marked up with position numbers, restriction sites, and feature labels. A line with amino acid translation of a selected sequence will also appear here once the translation has been executed with the Translate button. All elements in the Visualization Field are interactive. Moving mouse cursor over an element, clicking, or double-clicking it will trigger an action in most cases. The actions are listed in Table 1.
Table 1. Actions associated with elements of the Visualization Field
|Element||Mouse Cursor Over||Mouse Left Click||Mouse Left Double-click|
|Nucleotide Character (sense strand)||Displays position number||No action||Selects the position in the Editor Field|
|Restriction Site||Displays the cutting position on the sense strand and the total number of sites present||Displays a list of all cutting positions of the restriction enzyme||Selects the corresponding recognition sequence in the Editor Field|
|Feature Label||Displays name, type, and position of the feature||Selects the corresponding feature in the Feature Panel||Selects the corresponding sequence in the Editor Field|
|Amino Acid Symbol (in the translation line)||Displays a three-letter symbol and position of the amino acid in the translation||No action||Selects the corresponding coding triplet in the Editor Field|
Basic functions are performed with the buttons located under the Editor Field. These operations always include the entire DNA sequence.
Selection-related functions are executed with buttons located just above the Editor Field and are always applied to a selected part of the DNA sequence only.
Search Panel contains two input fields - one for entering a nucleotide sequence and the other for a restriction enzyme name. A search for a sequence can be done with both DNA and RNA sequences ('U's will be converted to 'T's). The sequence can contain numbers, spaces, hyphens, and other non-letter characters, all of which will be automatically removed. Letters other than A, C, T, G and U will be interpreted as degeneration symbols (if compliant with the IUPAC rules). All other alphabet characters will prevent any successful search. Although the sequence input field displays only one short line, a sequence of any length can be entered and searched for. It is also possible to search for a reverse complement of the sequence (click the Find Rev. Compl. button).
To check for the presence of recognition sites of a restriction enzyme, type or paste the enzyme name in the search field labeled "restriction site" and click the Find button. The restriction site search field is case sensitive, but not sensitive to space characters in the names so both "EcoRI" and "EcoR I" will be recognized, but "ecori" will be not. After performing a successful search for a sequence or a restriction site, yellow marked results will be displayed in the Visualization Field just below the search panel. You can click them to select the corresponding sequences in the Editor Field. Use a click followed by a Ctrl+click to select sequence ranges.
Feature Panel lists all the defined sequence features and provides management of their properties and visualization options. The handling of sequence features is described in detail in the part Sequence Features.
To open a file, choose the file with the Browse/Choose File button (the exact name of the button depends on particular browser and system local settings). Unformatted text (not MS Word!), Fasta and GenBank format sequences are supported.
To directly import a GenBank sequence, click the "download" icon located in the right upper part of the Main Panel. In the subsequent dialog, fill in the GenBank accession number and click the Download button.
To save a DNA sequence in a file, you have two options. Fasta format stores only the sequence labeled with a header (>name). To store a sequence with marked features, you have to use the GenBank format. This format was established for the DNA sequence records in the GenBank database. Due to this, it is supported by many if not all molecular genetics software products.
To work with multiple sequences in parallel (in silico molecular cloning), click the "new window" icon located in the center of the upper part of the Main Panel to open another WebDSV window.
You can combine uppercase and lowercase sequences in the Editor Field to keep track of changes you have made. The sequences in the Visualization Field will be all displayed in uppercase. Each time changes have been made in the Editor Field, the Apply & Process button has to be clicked to update the Visualization Field. In some browsers (e.g. Chrome) you can undo and redo recent changes made in the Editor Field by pressing Ctrl+Z and Ctrl+Y, respectively on the keyboard (the focus/cursor has to be in the Editor Field). To reverse all changes made in the editor field since the last use of Apply & Process, Update, or Map button click the Undo Changes button.
The Visualization Field cannot be used for editing. However, you can easily select a nucleotide position of interest in the Editor Field by double-clicking the corresponding nucleotide in the Visualization Field. Use a subsequent Ctrl+double-click on another position in the Visualization Field to select the intervening sequence. double-clicking an amino acid symbol in the translation line will select the corresponding encoding triplet in the Editor Field. Similarly, double-clicking a restriction enzyme name will select the corresponding recognition sequence in the Editor Field. All these selection actions will fix the position of the Main Panel and prevent its scrolling with the page. To switch the scrolling behavior of the Main Panel, click the anchor icon located in the lower-left corner of the Main Panel.
Nucleotide positions or ranges with an assigned function or marked for any other reason are called the sequence features. WebDSV enables the user to define sequence features in a few different ways with specific buttons (see Figure 2).
Figure 2. Ways to create new sequence features.
In all cases the feature will appear in the Feature Panel as a new item represented by a group of data fields and control elements. The function or meaning of each of the fields and elements is described in Figure 3.
Figure 3. Explanation of a sequence feature item properties, options and functions.
Any change or operation described further will get reflected in the Visualization Field only after clicking the Update button in the right upper corner of the Feature Panel. Accidental changes can be reversed with the Undo button located in the lower-right corner of the Feature Panel. This holds true only until the Update button has been clicked.
Visualizing too many features at once may produce a messy picture. You can change the visualization status ("on" or "off") of a feature by clicking the eye icon. Shift+clicking any eye icon in "on" or "off" state will switch all other eye icons to the same state. Ctrl+clicking any eye icon in "on" or "off" state will switch to the same state all other eye icons associated with features of the same type. To remove a feature, click the associated remove icon (white cross on red). Shift+clicking the icon will remove all features (confirmation will be required). Ctrl+clicking the icon will remove all features of the same type as the one being removed.
The eye icon has also a secondary function - a feature preview. When the mouse cursor is placed over an eye icon, the marked sequence of the associated feature in the Visualization Field will be brought to front (if overlapped by other features) and if any changes in its appearance has been made (but still not confirmed with the Update button), they will be displayed temporarily (until the mouse cursor is moved away from the eye icon). This is especially handy if you need to optimize the feature's color. To be able to see the preview, scroll the Visualization Field so that the feature is in view (you can use the "scroll to start" and "scroll to end" arrows located under the feature's name).
You can select the sequence of a feature in the Editor Field by clicking the ACGT button associated with the feature in the Feature Panel. Select a sequence range spanning two or more features by clicking the ACGT button of one item followed by Ctrl+clicking the ACGT button of another item. Moreover, you can use combinations of (double-)clicking and Ctrl+(double-)clicking in many other ways to select ranges of nucleotides, e.g. by double-clicking a position in the Visualization Field and Ctrl+clicking the ACGT button of a feature. In other words, any element the sequence of which can be selected in the Editor Field by a click (ACGT) or a double-click (all elements in the Visualization Field) can be combined with another such element to select the corresponding sequence range. The second click or double-click has to be done while pressing the Ctrl key on the keyboard.
Restriction sites displayed by default in the Visualization Field are a pre-defined selection of most frequently used enzymes. By default, all present sites are shown (this can be changed in Settings) and unique sites are shown in red (or yellow in the dark color scheme). The set of particular restriction sites to be searched for can be changed in the table invoked by clicking the dedicated icon located right to the Editor Field (see Figure 4A, B). The table offers a list of all commercially available restriction enzymes as offered by New England Biolabs, Thermo Fisher Scientific, Promega, Takara Clontech, Roboklon, Nippon Gene, Vivantis, and SibEnzyme.
Figure 4. Restriction site settings. A - Invocation of the restriction sites settings window. B - Appearance of the restriction sites settings window and its control elements.
The restriction enzyme selection can be changed either manually with checkboxes located in the last column of the table or by loading the restriction enzyme list from a file with the Load List button. The file should be a plain text file containing one restriction enzyme name per line. You can write such file in MS Notepad or similar program or by saving the current selection with the Save List button. The Reset button will restore the default selection list. The Set Permanent button will store the present selection in the browser memory. The success of this operation depends on the particular web browser and its settings. If successful, the selection will be preserved in all new browser tabs or windows in the course of the present session. It, however, might not be available in future sessions, so saving the selection as a file is highly recommended.
A presence of any restriction site can also be checked using the search panel under the Editor Field. To perform a more comprehensive restriction analysis, use the Restriction Analyzer tool.
To draw a map of features and restriction sites in the sequence, press the Map button located in the top part of the Feature Panel. If the browser asks you about allowing or blocking pop-up windows, choose "allow for this web site".
Figure 5. A map of features and restriction sites in the DNA sequence.
A map of features and restriction sites will appear in a pop-up window (if not blocked by the browser). Features and feature labels are interactive: After clicking a feature label, the corresponding feature item in the Feature Panel will get selected (see Figure 5 for an illustration). You can zoom the map with the slider located in the upper-left corner of the map window. The adjacent clickable text elements enable restoration of the initial zoom, automatic adjustment of the map window dimensions, and saving the map in a file - either as a PNG image or as a SVG file (scalable vector graphics). The latter can be further edited in programs like Inkscape or Adobe Illustrator.
By default, restrictions sites in maps are displayed with position numbers while features are without them. This can be modified in Settings. A few other options specific for circular sequences can be set. The diameter of the circle representing the DNA can be selected from three different sizes. The information depicted inside the circle can be switched between the sequence name or nucleotide position scale. Finally, the criteria by which adjacent restriction sites are grouped to list blocks can be changed.
WebDSV is a general purpose DNA sequence editor suitable also for the planning of molecular cloning experiments. In the easiest case - a replacement of a DNA fragment using the same restriction sites - just double-click the first restriction enzyme name in the Visualization Field, press Ctrl key and double-click the second restriction enzyme name. The corresponding DNA fragment will get selected in the Editor Field. Now you can use Ctrl+C and Ctrl+V keys for copying the fragment's sequence and pasting it into a target vector sequence already open in another WebDSV browser tab or window. In other cases, i.e. when using different but compatible sites (with the same overhang) or blunt ends, the exact start and end positions of the replacing fragment and in the destination vector have to be selected by the user. In these cases, double-clicking the nucleotide position in the Visualization Field rather than the restriction enzyme name will help to do the job.
WebDSV does not provide a fully automated PCR primer design. However, it facilitates manual PCR primer design. First, the real-time calculation of the GC-content and predicted melting temperature of a DNA fragment being selected helps to find a suitable primer-binding site. Second, marking the potential primer-binding site as a feature with the Forw. Primer or Rev. Primer button will display a link (the "PI" icon) in the primer feature item in the Feature Panel that upon a click will send the primer sequence into the Primer Inspector application where all potential primer dimers will be identified. You can send more primer sequences at once to Primer Inspector by first checking the checkboxes next to the primer feature items and then clicking the "PI" icon of any one of them.
A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics
SantaLucia, J. Jr.
Proc Natl Acad Sci U S A. 1998;95(4):1460-5.
Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas
von Ahsen, N., Wittwer, C.T., Schütz, E.
Clin Chem. 2001;47(11):1956-61.