Each issue of the DNASTAR newsletter will be featuring a short article written by a research scientist in the field of molecular biology. The intent of the article is to provide readers with additional information from their colleagues about work they are doing using sequencing as a key element. The first article is written by Professor Gabriel Dorado from Dep. Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain. For inquiries and additional information on the article, email DNASTAR.

Fig. 1. Search the Internet. Net Search allows researchers to interrogate different Internet databases from any Lasergene application module. In this case some of our contributions to GenBank are shown, including hits for prokaryotes, plants and animals. — Click to enlarge

Bioinformatics is associated with cutting-edge research, for which the Human Genome Project may be the key reference. We have contributed to such developments enhancing the DNA sequencing (Lario et al 1997, Anal Biochem 247: 30) and further avoiding false negative results with BLAST (Hernández et al 2000, DNA Seq 11: 339). But teaching should also be taken into account when considering the usefulness and potential of bioinformatics. To fulfill both research and teaching goals, a bioinformatics application needs to be accessible. Thus, it should be intuitive and easy to use. This allows even non-bioinformatics (ie. non-programmers) like most life science teachers and researchers to take full advantage of such tools. That was impossible in practical terms when only text-based consoles of command-driven interfaces were available only two decades ago.

Fortunately, with the development of the Graphical User Interface (GUI), and in particular the Mac paradigm on 24th January 1984, the dream of a bioinformatics package that is powerful, flexible and intuitive has become true.

Fig. 2. Discover and confirm SNPs.SeqMan built-in SNP search feature allows the automatic identification of mutations among DNA sequence data. The chromatograms displayed above the sequences greatly helps the SNP visualization and confirmation. — Click to enlarge

We have used different bioinformatics applications and packages for teaching and research at Universities and Research Institutes for the last 20 years. Amongst them, we have been gladly impressed with Lasergene for Mac OS first and Mac OS X now, because it excels in ease of use, and is also powerful and flexible. Most of the time it is not even required to look at the manual to exploit Lasergene’s possibilities, which is a good indication of the ease of use of any computer application.

Lasergene’s compatibility with other packages and between operating systems (Mac, Linux, Windows, etc) is a feature that we love. Additionally, the Internet accessibility to NCBI and other databases has been a tremendous bonus in recent years (Fig. 1). Actually, bioinformatics has never been the same since its integration with the Internet.

We have used bioinformatics in our research to help us clone (SeqBuilder) and sequence genes, find sequence identities via BLAST, ascertain open reading frames and further analyze genes (GeneQuest). Very useful also are the tools to reverse translate RNA into DNA and the translation of the latter into peptide, coupled with the identification of DNA and peptide motifs (Protean). We have also used PrimerSelect to design primers for hybridization, PCR amplification and sequencing of both ancient and modern DNA. The identification of SNPs and other mutations has allowed us to develop molecular markers to assist plant and animal breeding (Hernández et al 2001, Theor Appl Genet 102:1082), to diagnose diseases like osteoporosis (Quesada et al 2004, J Ster Biochem 90: 209) or to evaluate the environmental pollution (Cousinou et al 2000, Sci Tot Environ 247:213). Likewise, to identify individuals and population biodiversity and to allow food and feed traceability (“mad cow” disease), thus implementing fraud control. The SNP discovery is one of our main research goals, and for that the MegAlign and SeqMan Pro applications are invaluable tools, allowing comparison of several DNA sequences and chromatograms on the spot (Fig. 2).

Acknowledgements: supported by Grants AGL2006-12550-C02-01 & AGL2006-12550-C02-02 of ‘Ministerio de Educación y Ciencia’ (Spain).

Lasergene is a powerful DNA and protein sequencing software suite that allows users to perform many tasks easily. Each edition of our Newsletter will feature several different Tips and Tricks that should make your use of Lasergene even easier to use. Several of the Tricks that may assist you to perform certain tasks faster are listed below:

• Refining a Primer Search based on location
  
• Moving labels or Features on a Circular Map
  
• Setting default Assembly parameters in SeqMan
  

If you have particular Tricks for using Lasergene that you believe would benefit your colleagues, let us know so we can share them.

Refining a search for primers based on location?

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PrimerSelect easily allows you to specify a range to search for upper and lower primers.

To do this simply:

1. Launch PrimerSelect and enter your sequence by going to File»Enter Sequence.

2. Then, go to Conditions»Primer Locations

3. From the Restrict Locations drop-down box, select “Upper and Lower Primer Ranges”

Type in ranges for your upper and lower primers, and then click OK.

Want to move the labels or features around on your circular map?

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SeqBuilder allows you to easily move features, enzyme labels, and the diameter of the circle just by clicking and dragging.

To do this:

1. Double-click the item you want to move (feature, enzyme label, etc.).

2. Click and drag the item where you want it.

1. Select the feature by double-clicking it.

2. Right-click on the feature and select Detach Feature Label. The detached feature labels can then be moved around as described above.

Tired of changing your assembly parameters for each project?

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Setting default parameters is easy!

1. First, launch SeqMan.

2. Then, close the Unassembled Sequences and Untitled project windows.

3. Go to Project»Parameters.

4. Adjust any of the parameters listed to your preferred settings, then click OK.

That’s it! The settings you have chosen will now be your default for all future projects. And, they can easily be changed again by repeating the process above.

SeqBuilder has the ability to do Automated Virtual Cloning for TA, TOPO®, Gateway®, and restriction site protocols. Restriction cloning is a general cloning method that utilizes restriction enzymes to cut insert and vector sequences at specific locations. Once vector and insert sequences are properly prepared, a ligation reaction is typically used to create a recombinant clone.

1. Launch SeqBuilder.

2. Select File»New Cloning Project. The Untitled Cloning Project window (pictured below) appears.

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3. Add your Insert and Vector sequences to their respective folder by right clicking on each folder and selecting Import.

Note: You may also import vector sequences from the standard DNASTAR vector catalog by selecting File»Open and locating CloningVector.sbp in:
C:\Program Files\DNASTAR\Lasergene on Windows platform or Applications:DNASTAR:Lasergene on Apple Macintosh platform. Once the CloningVector.sbp project is open, you can copy and paste vectors from the catalog into your Vectors folder.

4. Open your Insert sequence by double-clicking it from the Inserts folder.

5. Display your enzymes by opening the Enzymes Displayed folder, and either selecting the individual enzymes, or selecting filtered group, such as all Unique Sites.

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6. Choose the enzymes to use for restriction cloning by clicking on an enzyme label and highlighting the label and the nucleotides between the two cut sites.

7. Select Cloning»Copy Restriction Insert. The insert is copied to the clipboard.

8. Next, open your Vector sequence from the Vectors folder.

9. Select the restriction enzyme sites on the vector to clone into by clicking on an enzyme label and highlighting the label and the nucleotides between the two sites.

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10. Select Cloning»Clone Restriction Insert. The Clone Insert dialog (shown on the right) opens displaying the ends of the vector and insert sequence for compatibility. If ends are not compatible, left end and right end checkmarks at the top will be unchecked (Windows only), and the Clone button will be inactive.

11. Modify the ends, if needed, by sliding the positional arrows or by clicking Fill in or Trim Blunt. When both ends are compatible, both right and left end checkboxes will be checked (Windows only), and the Clone button will be active.

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12. Enter a name for the new clone in the Save As Copy titled box, and save to any project listed in the in project box.

13. Click Clone. The new clone is displayed in a new window, and it is stored in the Clones folder of that Project.