Restriction-enzyme cut sites

Mapping of restriction sites is started by clicking the scissors button . Analysis opens in the multi-purpose pane. Which enzymes are used in scanning can be filtered by selecting categories in the 'Enzyme filter'. Recognition sequences 6 nt or longer and with 5' overhang are included in analysis by default. Restriction enzymes that have too many cut sites may be filtered by setting the 'Max. cuts' value ("show only enzymes that cut this or fewer times"). A Max. cuts value set to zero will list enzymes that do not cut. When set to 1 or higher, zero-cutters are not displayed. To apply new filter settings to an open analysis, click the button again.

The calculator button is used to display a report of the fragments that would result in an enzyme digest, based on selected rows/enzymes. The report is displayed in the Text Editor and can be saved to file or copied to a document. Restriction-enzyme functions take into account the linear/circular status of the sequence.

Cut sites are annotated in the usual way; select rows/enzymes and click the annotate button . The text label on annotated restriction-enzyme cut sites should not be modified. The text is used to look up enzyme properties, like overhang type and length, which is needed to position cut sites correctly when strands are switched (reverse complement).

Open reading frames

Open reading-frame mapping is started by clicking the roadmap button . Analysis opens in the multi-purpose pane. The ORF> text box is used to set the minimum length in nucleotides, the number is edited directly by keyboard input. The linear/circular status is accounted for in analysis. Open reading frames are annotated in the usual way; select rows/ORFs and click the annotate button .

Translation to protein

Translation to protein is started in the sequence pane's right-click menu (or the Analysis menu). Before starting analysis make sure the open reading frame appears in forward direction. Switch strands (reverse complement) if necessary. Either select a subsequence in the sequence pane, or position the cursor just in front of the ATG start codon, or any codon of choice, and click on a "Translate to protein" menu item. If a subsequence is selected, translation will be confined to the selected region. If nothing is selected, translation will start at cursor position and proceed to the first in-frame stop codon. The resulting protein sequence opens up in a Text Editor window, from which it can be saved to file or copied to another document or open project.

Codon-usage compatibility analysis

Codon-usage compatibility analysis is started in the sequence pane's right-click menu (or the Analysis menu). Before starting analysis make sure the open reading frame appears in forward direction (switch strands if necessary) and place the cursor just in front of the ATG start codon, or any codon of choice. The function will analyse the given open reading frame and output statistics on actual codon usage for the different codon triplets and a comparison to normal codon usage and what would be the optimal numbers. The results are displayed in a Text Editor and may be saved to file or copied to a document.

Create optimized open reading frame (ORF)

Create optimized open reading frame (ORF) sequence for expression in a selected species. The source sequence may be DNA, RNA or protein. The optimized ORF is assembled from scratch, using codon triplets according to the species' codon usage frequencies. Before starting analysis make sure the open reading frame appears in forward direction (switch strands if necessary), and place the cursor just in front of the ATG start codon, or any codon of choice, or select a subsequence.

Several modifications may be applied:

Reduce rGTP contents: skew codon usage away from rGTP-containing codons for better in-vitro transcription with Cap nucleotides, in which a low rGTP concentration is the limiting component for yield.
Reduce GC contents: skew codon usage away from rGTP- and rCTP-containing codons to reduce probability of secondary structures in single-stranded RNA/DNA. This option overrides the 'Reduce rGTP..' setting.
Exclude rarely used codons. A cut-off value may be set between 1-9 %. When expressing proteins at moderate levels alongside all other active genes in a cell, it may be a good idea to skip low-frequency codons, since the consumption of tRNA-AA by the ectopically expressed gene/mRNA is negligible compared to the total consumption, and the gene/mRNA will then benefit from using high-frequency codons. A different situation is protein production in a lysate. To maximize protein yield one must make use of all the tRNA-AA present, in which case it's best to use all codon triplets according to the normal codon usage frequencies in the species.
Add a stop-codon if missing: which may be relevant if the source sequence is protein or a selected subsequence.

Oligo primer analysis

Oligo primer analysis is started by clicking the primer button . The analysis is based on the nearest-neighbor free-energy model and the unified thermodynamic values published by SantaLucia (John SantaLucia Jr. (1998). "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics". Proc. Natl. Acad. Sci. USA 95 (4): 1460–5). Many primer analysis programs use old and outdated thermodynamic data, for backward compatibility, since many are used to the old dG values. However, the SantaLucia tables are usually recommended. It's a matter of getting used to the new dG values, which are approximately half of the old ones, on average. The benefit is having more accurate results.

The primer analysis pane has many adjustable parameters and two multi-column tables and will benefit from having enough space. On most PCs the PlasmidTools window should be maximized to the entire screen when running primer analysis.

If a subsequence is selected in the sequence pane when starting analysis, primer search ranges are initially confined to the adjacent 100 bp on each side of the selection. If nothing is selected, primer search ranges are set at the beginning and end of project sequence. Search ranges are of course adjustable.

The first thing to do after opening primer analysis is to select analysis mode, sequencing or PCR. Whenever the mode is switched the parameters are replaced by default values, which are the common values in use. The search ranges are not modified.

Primer sequences are annotated in the usual way; select a row/primer in forward and/or reverse direction and click the annotate button Annotate icon .

The primer melting temperature, Tm, is the temperature at which 50% of the sites to be primed are bound by primer. The temperature used in PCR and Sanger sequencing should be 3-5 degrees below this for optimal priming.

A primer-dimer dG value of '99', with no decimals, means that no di-nucleotide interaction was found, and thus, the lowest affinity possible. Cross-dimer dG values are only shown in PCR mode.

A detailed report on selected primers, including textual drawings of primer-dimer interactions, can be opened from the right-click menu ('Primer report'). The report is dispayed in a Text Editor window, from which it can be saved to file or copied to a document.

The default parameter values will not always fit perfectly. What is suitable values depends on the nature of the sequence, for example if it's rich in GC or not. The default values are set quite wide, or not to stringent, which is a good starting point. The exception is the 3' dG, which is a critical parameter for obtaining primers with high specificity. If no or too many primers are found, first try to adjust primer length, max run, or primer Tm settings, whichever fits best. The 3' dG values should not be modifided, unless there is a good reason to modify it.

Map hairpin structures

Hairpin (stem-loop) structures may form in single-stranded RNA/DNA despite of distance between complementary sequences. This function maps complementary sequences present in project sequence, or selected subsequence, that are separated by at least three nucleotides. Stem-length range and minimum Tm are adjustable parameters. Results are listed in a table and selected row is higlighted in the sequence pane and may be annotated the usual way .

Sequence search

Search for subsequences is done by clicking the search button , which opens up in the multi-purpose pane. Then enter or paste a sequence in the text box and click the adjacent search button. Although the text box is small, the sequence can have any length. A minimum percentage identity threshold may be set. Matching sequences are displayed in the multi-purpose pane. Search results are annotated in the usual way, select rows/matching sequences and click the annotate button .