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!

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.

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

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA – Compare tab and select the Internet – Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is used for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.
NCBI BLAST is set as the default URL.

Fig4. Internet Blast Search Parameterset dialog box.

5. Press the Setting… button to edit the parameters of NCBI BLAST.

Fig5.　NCBI Advanced BLAST Search dialog.

6. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

7. Press the Execute button to send the query.
The time it takes to retrieve your results will depend on the size of the sequence, the database, and your internet service provider.  Once your results come back, it will be displayed like in Fig6.

Fig6. A sample result of the BLAST search in DNASIS MAX.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA - Compare tab and select the Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.

Fig4. BlastParameters dialog box.

5. Select the Blast program type from the Program name box.

• blastp – compares an amino acid query sequence against a protein sequence database
• blastn – compares a nucleotide query sequence against a nucleotide sequence database
• blastx – compares a nucleotide query sequence translated in all reading frames against a protein sequence database
• tblastn – compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames
• tblastx – compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

6. Set the E-value or Expect value in the Expectation value field. This value represents the number of times you would expect to see such a match (or better) merely by chance. The closer the value is to zero, the less likely the event is. To re-iterate, the smaller the E-value, the more significant the match is and the lower number of hits you’ll see.  The default value is 10.

7. Select the intended database from the Nucleotide Database.
Select the In-house-NA for this tutorial.

8. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

9. Press the Execute button to start the blast analysis.

Fig5. A sample result of the Blast search in DNASIS MAX.

For more information on DNASIS MAX, please visit the DNASIS MAX product page or download the FREE trial.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the

C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA – Search tab and select the Primer Design option.

Fig2. DNA – Search tab.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, there are the Parameter and Execute buttons.  The Parameter button is for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.

Fig4. PrimerParameterEditor dialog box.

5. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

6. Press the Execute button to start the primer design.

Fig5. A sample result of primer design in DNASIS MAX.

For more information on DNASIS MAX, please visit the DNASIS MAX product page or download the FREE trial.

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

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.

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”

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!

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.

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.

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

1. You can now export individual results (summaries or raw) from your lab notebook as PDF files or print them out.

2. The PrimerX tool has been added to support primer design for mutagenesis. It even works with multiple sequences!

3. You can now extract email addresses from your GMail or Yahoo mail accounts to make it easier to invite your colleagues to collaborate with you through DNASIS SmartNote.

4. If you find yourself using the same tools frequently, you’ll love this feature: when it’s time to pick a tool for an analysis, you can now see the 5 most recently used tools listed at the very top and just click them to quickly launch the analysis.

Coming soon… an exciting new way to share data with colleagues!

1. By popular demand, you can now import GenBank and multi-GenBank files into SmartNote, including those exported from other programs, such as DNASIS Max and Vector NTI.

2. We have added a new option for displaying the predicted amino acid sequence aligned with a DNA sequence. Use the ExPASy Translate tool and specify “Includes Nucleotide Sequence” for the “Output Format” option. Thank you, Jessica Min for this suggestion. This should help users who need to design primers for mutagenesis.

3. Support for IUPAC codes in sequences is also being added this week.

Please keep the feedback coming!