This guide supports the Galter Library class called Nucleotide Sequence and Structure. See our Classes schedule for the next available offering. If this class is not on our upcoming schedule, it is still available to you or your group by request.
This class is based on the excellent NCBI classes formerly offered as the NCBI Advanced Workshop for Bioinformatics Information Specialists. Specifically, the modules DNA Analysis, taught by David Osterbur of Countway Library at Harvard University; and RNA Analysis, developed by Nicola Gaedeke of Biotools.info, Rana C. Morris of the NCBI, Donna Messersmith of LABS-Now LLC, and Kristi Holmes of Becker Library at Washington University at St. Louis.
The best way to find a nucleotide sequence is to begin with NCBI's GenBank, which is searched through the NCBI Nucleotide database.
Go to the NCBI home page:
You can begin your search simply by typing search terms in the search box. You can use boolean operators (AND, OR, NOT) to create a more focused search by adding organism names, keywords, gene names, etc. This guide uses the example of early growth response 1. Type EGR1 in the search box and search.
Now click the results in the category Nucleotide.
If you see the sequence you are searching for easily in your results, you can click the description to access the record. However, many sequences are returned in Nucleotide database results. To narrow your search within Entrez Nucleotide, you can use many of the features available on the nucleotide results page.
Click on the blue link for the number of results in Gene (152 in this example).
Scroll down the results in Entrez Gene until you see the record for the entry you are seeking. Species are listed in square brackets in the descriptions for each gene entry.
Click the blue gene symbol link to access the detailed record.
On the Entrez Gene record for your selected gene, you can look in the right side blue Links menu for the reference sequences for genes or mRNAs using the RefSeq links.
To access the best curated reference sequence for your gene, scroll down the page to the section labeled Genomic regions, transcripts and products.
In this area are
Click on Try our new Sequence Viewer.
Sequence Viewer allows you to view and link to many features of your genomic or RNA sequence from a single interface. You can
Right click on the green (genomic view) bar, move your mouse down to Views & Tools, and click on the FASTA link. Your record will open in a new window. Only the sequence for your gene's region is displayed in the chromosome assembly record.
You can change the region shown or display the entire chromosome cDNA sequence.
Copy the FASTA format of the cDNA from this page.
There are many web servers that provide restriction maps for selection of enzymes to cut DNA sequences. Unfortunately, many of these sites are not updated to reflect currently enzyme availability, and are therefore not listed in this guide. Below are some restriction mapping servers that are available. Users are advised to try a few of the available restriction mapping websites to determine which suit their needs best.
NEBcutter is provided by New England BioLabs and reflects the current available enzymes from their catalog, but allows users to explore restriction enzymes supplied by all commercial suppliers and some non-commercial suppliers, as well. NEBcutter can be found at the URL:
Your restriction map will be returned in a graphical format, with the names of the restriction enzymes as clickable links to their available vendors' pages.
RestrictionMapper can be found at the URL:
This site is updated approximately yearly to include all enzymes in the REBASE restriction enzyme database, so it is more current than some restriction mapping sites.
RestrictionMapper does not return a graphical map of restriction sites, but the table of results is easy to read and includes links to suppliers of each restriction enzyme.
Other Restriction Mapping Websites
As with restriction mapping, there are a number of Web servers that will generate primers for you for a nucleotide sequence. Selection of primer pairs for your sequence is important, because you don't want to create primer dimers: primer pairs that will bind to each other instead of binding to your nucleotide strands.
NCBI's Primer-BLAST is based on the Primer3 software program developed at the Whitehead Institute and the Howard Hughes Medical Institute. Primer BLAST provides users the convenience and speed of using NCBI's BLAST servers to generate primers from their sequences.
There are a couple of ways to access Primer-BLAST at the NCBI:
After you click the Get Primers button:
Users who wish to know more about Primer3's design and use are encouraged to consult the Open Source manuscript:
Steve Rozen and Helen J. Skaletsky (2000). Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386.
RNA can take on many complex three-dimensional structures, since it is (usually) a single stranded molecule and thus more flexible than DNA. To find nucleotide structures in the NCBI Structure database (MMDB), use the Limits or Preview/Index tabs to focus your search of structures on RNA or DNA.
NCBI's Structure Database, the Molecular Modeling Database (MMDB)
Go to the NCBI's Structure database home page:
Example: You want to see any structures in the database that include 2 chains of tRNA.
Your results will consist of any structures that have 2 tRNA chains.
The Nucleic Acid Database (NDB)
If you want to search in a database that contains only nucleic acid structures, the NDB is a good option.
The NDB's home page:
Select Search on the NDB home page.
The search page has numerous fields that will help you narrow your search to find if any NMR or x-ray crystallography structures exist for your nucleotide of interest.
NDB also is home to a number of other tools and resources:
Other Nucleotide Structure Sites
Many other good nucleotide structure sites are available on the Web.
Other Useful Nucleotide Sites (Miscellaneous)