An online social community for nearly every niche exists on the internet.  MiraiBio understands this so now, there is one for the Luminex Community, MasterPlex Community, and DNASIS Community.

In addition to the online community members, MiraiBio’s own employees will be browsing around as well joining in on all the conversations.  So whether you were just curious about something or need help troubleshooting, feel free to drop by and start a converation!

DNASIS SmartNote is a web application that is capable of doing what a stand-alone desktop software does BUT inside a browser.  With this capability comes some requirements needed to power the web application.  Here is a list of some requirements as well as some recommendations to optimize your DNASIS SmartNote experience:

1. Web browser - The latest version of FireFox (v3) is highly recommended for a much faster usage of DNASIS SmartNote.
2. JavaScript – JavaScript is required in using DNASIS SmartNote.  If you are having issues loading up or viewing the application, your JavaScript may be disabled.  Here are some instructions for enabling JavaScript in different browsers.
3. Screen Resolution – A minimum screen resolution of 1024 X 768 is recommended although I would use 1280 X 1024 or higher if possible.
4. Avoid cramming – Avoid running all your analyses on one single page.  This will slow down the loading of your page.  Instead, create new pages by clicking on the Add Page link on the top of the left navigation tree.

DNASIS MAX has the ability to search for homologous sequences to your sequence with BLAST (Basic Local Alignment Search Tool). BLAST finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.

The following procedure guides you through utilizing BLAST against NCBI’s sequence databases.

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DNASIS MAX has the ability to search for homologous sequences to your sequence with Blast which is
useful in judging if the sequence is novel or even add annotations from known gene information. The following procedure guides you through utilizing Blast against an internal sequence database.

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DNASIS MAX uses the Primer3 engine for designing primers, which is one of the most frequently used procedures in daily research.  The following procedure guides you through designing primers utilizing the DNASIS MAX interface.

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1. DNASIS SmartNote now shows a graphical view of any sequence annotations included in files imported from Entrez. To see it, click the “Details” link next to the sequence in the “My Sequences” view. Here’s what it looks like:

2. The xTAG Tool now lets you exclude certain bead regions. In addition, its results now also include tag and anti-tag sequences.

NCBI BLAST, the Basic Local Alignment Search Tool (BLAST) is a suite of programs designed to search all available sequence databases for similarities between a protein or DNA query and known sequences. BLAST allows quick matching of near and distant sequence relationships, providing scores that allow the user to distinguish real matches from background hits with a high degree of statistical accuracy.

Focusing on local alignments, BLAST uses a heuristic algorithm to detect relationships between sequences that may only share isolated regions of similarity. BLAST results take sequence length and the nucleotide/peptide compositions of the query into account when assigning alignment scores. For sequences shorter than 200 residues, an effective length is used to compensate for “edge effects”. Sequence alignment scores are reported by BLAST programs as E-values that reflect the strength of alignment between a given sequence in the database and a query. E-values are reported instead of the traditional P-value, to improve resolution between low scoring alignments, but for closely related sequences (P < 0.01), these values are nearly equal.

For more detailed information on how BLAST scores are calculated, visit:

http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html

For most first-time users of BLAST, choosing the right sub-program may be difficult. BLAST offers a variety of search tools for different types of queries. In general, the best choice of program depends upon the sequence length, the database being searched, and the information requested in the search.

Nucleotide BLAST is a collection of programs allowing users to compare a query sequence against other nucleotides in the database. BLAST accepts sequences in a variety of formats, including FASTA, GenBank, and Accession/GI numbers, and compares these with the NCBI databases. MEGABLAST is a concatenating algorithm for quickly aligning sequences longer than 28 residues. For shorter sequences, such as primers, standard nucleotide-nucleotide BLAST offers automatic parameter settings suited to these queries.

Protein BLAST is a collection of programs used to find protein sequences similar to a query. These programs accept sequences in the same file formats as Nucleotide BLAST. PSI-BLAST is a position specific, iterating algorithm that searches sequences from each round as the basis for scoring sequences searched in the next round. It distinguishes between highly and weakly conserved positions in the sequence, resulting in increased sensitivity with each iteration. PSI BLAST also offers the option of including regular expression patterns in the search, allowing users to identify sequences that include a pattern and are homologous to the query protein sequence. As with Nucleotide BLAST, Protein BLAST includes automatic parameter settings for shorter sequences.

Translating BLAST operates in a similar fashion to both the nucleotide and protein search routines. BLASTX translates nucleotide sequences into protein sequences in each of the 6 reading frames, prior to comparing the query to the protein databases. TBLASTN compares a protein sequence query against a database of nucleotide sequences previously translated in each of the 6 reading frames.

Users can refer to the NCBI BLAST program selection guide for more information:

http://www.ncbi.nlm.nih.gov/blast/producttable.shtml.

Users can access BLAST tools directly through the web, or through a variety of software applications, such as MiraiBio’s DNASIS SmartNote, which helps users find and organize sequences, and automatically submit them to the BLAST programs. DNASIS SmartNote has the additional ability to BLAST multiple sequences “in batch” without tediously copying/pasting each sequence and waiting for each result to come back.

To learn more about DNASIS SmartNote, visit http://smartnote.miraibio.com.

We have 3 improvements to report for DNASIS SmartNote this week, all having to do with more efficient sequence importing and exporting:

1. Accession number recognition – DNASIS SmartNote now recognizes accession numbers in sequences you import (or will ask you to enter one if you paste in a raw sequence). This gives you better control over the naming of your sequences, so you can keep better track of which results belong to which sequence.

2. Import multiple sequences at once – If you’d like to import several sequences into DNASIS SmartNote and you know their accession numbers, you can now enter them all (separated by commas). To try it out, click on “Get Sequences” under DNASIS SmartNote’s “Sequences” tab, then select “Search for sequences from NCBI Entrez” or “Search for SNPs from NCBI DB”

3. Export raw results from xTAG Software tool – For those who are using DNASIS SmartNote’s new tools for designing tagged, gene-specific oligos (GSO) for their Luminex assays using Universal Array beads, the xTAG tool can now export results as comma-separated values (CSV), so you can transfer the data to Microsoft Excel or other spreadsheet programs. The output fields are: “Sequence Name”, “Bead Region” and “Oligo Sequence”

We’ve added a new “Friends” tab to make it easier to invite friends and colleagues to use DNASIS SmartNote. There are two options:

1 – Invite Friends – This is just an easy way to tell your friends and colleagues about DNASIS SmartNote, because every molecular biologist should be using it.

2 – Invite and Share Notebook with Friends – This lets you not just invite friends to get their own account, but also to share their notebook and sequences with you. If they agree, you’ll be able to collaborate in cool new ways. First, you’ll be able to see read-only views of each other’s notebooks, which will appear below your own notebook navigator in DNASIS SmartNote’s left pane. Second, you’ll also be able to easily view or import each other’s sequences. You can then run your own analyses on your friend’s sequences. How cool is that?

On a separate “note”, we’ve added a new option under the “Sequences” tab for importing SNP sequences from NCBI. You can either search by keyword or by accession number. Enjoy!

1. A job queuing system was implemented – You no longer need to wait for compute-intensive results (e.g. BLAST searches on multiple sequences) to come back. Just submit a job and continue working. When the results are in, you’ll be notified.

2. You can now add comments to tools and read other users’ comments. See what your colleagues have to say about which tools are best for a given task, or if there’s any tips and tricks to get the most value from a tool.

3. Auto-scrolling to results has been fixed – When you click on a result in the left pane, the right pane will automatically scroll to the corresponding position on the page so you can instantly access any result.

4. More help features to guide new users – You’ll notice several new icons to help you use SmartNote’s features.

5. Other cool additions such as the new animated progress icon for the sequence manager window.

6. Many bug fixes – everything just works more smoothly.

Remember to tell us what you think about DNASIS SmartNote and what new features you’d like to see. We’ve been making changes weekly, so as long as your request is reasonable, we’ll most likely be able to add it.

By default, DNASIS SmartNote will track PubMed articles related to up to 10 of your most recently imported sequences. You can view these regularly updated articles by clicking on the “Articles” tab. From here, you can also clip individual articles into your notebook, as well as print and email them.

If you want to receive regular email updates for new articles, you’ll need to explicitly mark at least one of your sequences listed in the “My Seq’s” tab. You will then start receiving regular emails with any newly published articles related to your sequences. Since you can only track 10 sequences, the sequences you have explicitly marked will get higher priority.

How does DNASIS SmartNote find related articles? If the sequence was imported as a GenBank file, DNASIS SmartNote looks for gene names in the annotations. Otherwise, it defaults to a plain text search of words in the sequence’s description.

We invite you to try out this new feature and let us know if we can do anything to improve it for you.

Since PCR primer design is one of the most widely used features of DNASIS SmartNote, we did some research and put together a list of the top 10 tips for designing PCR primers that work. When designing oligonucleotide primers for PCR, it is helpful to keep some considerations in mind to optimize the output and specificity of your experiment. Here are some tips gathered from experts to get you started:

1. Design your PCR primers to be 18-30 oligo nucleotides in length. The longer end of this range allows higher specificity and gives you space to add restriction enzyme sites to the primer end for cloning.

2. Make sure the melting temperature (Tm) of the primers used are not more than 5°C different from each other. You can calculate Tm with this formula: Tm = 4(G + C) + 2(A + T)°C

3. Aim for a Tm between 65 and 70°C for each primer over the region of hybridization

4. Use an annealing temperature (Ta) of 10 to 15°C lower than the Tm.

5. The GC content of each primer should be in the range of 40-60% for optimum PCR efficiency.

6. Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming.

7. Avoid long runs of the same nucleotide.

8. Check that primers are not self-complementary or complementary to the other primer in the reaction mixture, as this will encourage formation of hairpins and primer dimers and will compete with the template for the use of primer and reagent.

9. If you can, make the 3′ end terminate in C or A, as the 3′ is the end which extends and neither the C or A nucleotide wobbles. This will increases the specificity.

10. You can avoid mispriming by making the 3′ end slightly AT rich.

11. Use the right software. OK, so it’s 11 tips. Using the right software is a great way to automate these steps and minimize errors, especially when you have to design primers for many sequences. DNASIS SmartNote includes several primer design tools and is also a lab notebook that automatically keeps a record of your analysis results. You should definitely give it a try. Click here to sign up a free account.

If you prefer traditional desktop software, take a look at DNASIS Max instead.

References:

http://rothlab.ucdavis.edu/protocols/PrimerDesign.html

http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/oligo.html

http://www.protocol-online.org/prot/Molecular_Biology/PCR/PCR_Primer/

http://www.mcb.uct.ac.za//pcroptim.htm