In order to expose you to a variety of parameters, there are two practicals built into this short tutorial. The first one will look at creation of a restriction map for a DNA sequence. The second one is designed to create a multiple sequence alignment from three protein files. Each tutorial can be completed independently of the other one.

In order to complete this practical your computer must be connected to the internet, as the software retrieves database entries from the sequence repositories at the European Bioinformatics Institute, UK.

Restriction Mapping

Access the Display Restriction Sites option from the Favourites menu in Jemboss. Type embl:AF361440 into the Sequence Filename box and hit the LOAD SEQEUNCE ATTRIBUTES button.

This selection causes the restriction mapping program remap to be loaded into the Jemboss program form. remap offers a variety of restriction options and allows the translation of the sequence to be displayed. Your software can read input sequences directly from the database using the USA syntax. Alternatively, if you already have a sequence, it can be dropped and dragged from the File Manager into the input field or cut and pasted from elsewhere using the paste selection and the command ctl V (hold down both the control key and the V key at the same time).

The LOAD SEQUENCE ATTRIBUTES button ensures that the program determines whether the sequence is nucleic or peptide and calculates any default parameters. In this case only a nucleotide sequence is acceptable. Whilst there would be no error message on loading the attributes for a protein sequence, the warning would appear when the program was run.

This sequence is the full genomic DNA sequence of the pBT cloning vector. At 3210bp long, the resulting display would be too long, so we are only going to display the region around the multiple cloning site.

Click on the Input Sequence Options button and type 2330 into the begin field and 2409 in the end field. Hit the OK button to close the window.

In accordance with many other computer programs, only 60 characters will be shown on one line at once by default. However, remap allows the user to define the width of display, which will be done further down the program form.

Alter the minimum recognition site length by clicking once in the text field, deleting the default selection of 4 and replacing it with 6.

Scroll down to the Output Section and uncheck the boxes for Display Translation and Display cut sites and translation of reverse sense.

The lengths of restriction sites for restriction enzymes vary, although 6 cutters are possibly the most common. The lower the value in this box, the more enzymes will be visible on the final results display. The EMBOSS application includes ambiguity matches for a restriction site within the 6 base recognition sequence.

This restriction programme will display protein translation of the nucleotide sequence of both the forward and the reverse sense. These may complicate the display if you are confident of the direction of your sequence.

Type 2342-2397 into the Regions to put in uppercase. Alter the Width of sequence to display to 80 by clicking once with the cursor in the edit box and altering the default setting. Leave everything else as default.

The display width will be altered to accomodate 80 characters to allow us to see several residues either side of our multiple cloning site. The exact MSC will be displayed in upper case to make it readily identifiable.

Many of the programs in your BioBind suite have a variety of options that are crucial to the program and allow it to run. There are also a variety of additional options and some rather more advance options. These are all available on the program form.

Click the Advanced Options button at the bottom of the program form. Scroll down and check the box to Force single site only cuts to ensure that only unique sites are displayed. Uncheck the box to disallow Allow ambiguous matches.

There are several further options for this application. The options to select genetic code and translation frames do not apply in this case, as the results display of our selections does not involve translation of the nucleotide sequence.

Hit the GO button and the results window will automatically pop up in the browser.

The unique restriction enzymes with unambiguous 6 base recognition sites are displayed along the length of our selected 80 bases within the correct strand of the sequence. Any enzyme isoschizomers are listed below the initial display.

Further information on the restriction enzymes are displayed on the results by default. These include the enzymes that cut less than the minimum selection [default 1] and more than the maximum selection [default 2,000,000,000]. As we forced single cut sites the default minimum is reduced to 1 also and the two enzymes that are displayed represent those that cut within our 80 base region but do so more than once.

It also reports the enzymes in the Rebase database which match all parameters but that do not cut in this sequence and those are neither blunt or sticky cutters, nor contain ambiguous recognition sites, or are commmercial enzymes.

Multiple Sequence Alignment

Select Multiple Alignments from the Favourites list. Select the list of files.

in the first one type swissprot:calm_human.

In the second type swissprot:calm:mouse.

In the third type calm_rat.

Hit the LOAD SEQUENCE ATTRIBUTES button.

The list of files will enable upto 20 different files to be entered. These files could be anything from an entered USA (as above) to single and multiple sequence files or list files. Files may be entered in any format that is supported by EMBOSS.

The LOAD SEQUENCE ATTRIBUTES button ensures that the program determines whether the sequences are nucleic or peptide and calculates any default parameters.

There are very few options which are crucial to the launch of emma once the sequences have been introduced. They concentrate on options relating to the dendrogram file that is produced by the program to define how it clusters the individual sequences to produce the alignment. In this tutorial, we will look at the entire alignment.

Hit the Advanced Options button and scroll down to see the first of these additonal options. Uncheck the box Do you wish to carry out a fast or slow pairwise alignment to carry out a fast alignment.

The default (checked) is a slow pairwise alignment of each individual sequence entered into the programme. In this case, there are 3 input sequences so there would be 3 pairwise alignments and a slow alignment would be perfectly feasible, but we will select the fast alignment. As this option is unchecked, parameters that are no longer available are greyed out.

As the matrix can no longer be selected for the pairwise alignment, the program will use the default which is a series of BLOSUM matrices to create the optimal alignments.

Generally it is difficult to design parameters for an initial alignment, so we will accept all other options as their default.

Scroll back and hit GO to launch the program. Hit the Show Alignment button to view the alignment created.

If you launched Jemboss from a web page, then you will have to save the alignment first. Hit the Job Manager bar to display the results. Highlight the result and Display this file. Select the aln tab and save the result. Highlight this file in the local file manager and right click to access the menu. Open with the Jemboss Alignment Editor.

In this case the sequences are identical and so there is no adjustment needed regarding the multiple alignment parameters. Should your selected sequences appear differently, there is the option to edit the alignment in the Jemboss Alignment Editor or return to the multiple alignment file and alter some of the parameters.