Nucleotide BLAST

Nucleotide BLAST refers to the use of a member of the BLAST suite of programs, such as “blastn” to search with a nucleotide “query” against a database of nucleotide “subject” sequences.


Available Nucleotide-Level Searches

There are two members of the BLAST suite of programs that are designed to make nucleotide-to-nucleotide alignments. The first is the original BLAST nucleotide search program known as “blastn.” The “blastn” program is a general purpose nucleotide search and alignment program that is sensitive and can be used to align tRNA or rRNA sequences as well as mRNA or genomic DNA sequences containing a mix of coding and noncoding regions. A more recently developed nucleotide-level BLAST program called MegaBLAST (7) is about 10 times faster than “blastn” but is designed to align sequences that are nearly identical, differing by only a few percent from one another. MegaBLAST allows the rapid mapping of a transcript onto a typical 3 billion base mammalian genome in seconds, and is useful for processing large batches of sequences. A refinement of MegaBLAST, known as discontiguous MegaBLAST, uses a discontiguous template to define an initial “word” in which characters in some positions, such as those in the wobble base position of codons, need not match. Discontiguous MegaBLAST allows rapid cross-species mappings involving coding regions in cases where species differences in codon usage would prevent alignments using the original MegaBLAST program.



Examples of Nucleotide BLAST Searches

Problem 1

Click on the link indicated by “P” next to the “Nucleotide-nucleotide BLAST (blastn)” to access the problem. This problem demonstrates how to use BLAST to find human sequences in GenBank that can be amplified with a particular primer pair. Access the nucleotide–nucleotide BLAST page (by clicking on the Nucleotide–nucleotide BLAST link). Paste both the forward and reverse primers into the BLAST input box. Insert a string of about 30 N’s after the first primer sequence to separate the two sequences to be found in separate, not overlapping alignments. Limit your search to human sequences by selecting “Homo sapiens” from the “All organisms” pull down menu under the Options for advanced blasting and click the BLAST! link. Retrieve results by clicking on the “Format” button. Look for two hits to the same database sequence.

In this result, shown in Fig. 1, there are 13 GenBank entries that align to both the forward and reverse primers at different locations (indicated by thick bars) with a gap in between (indicated by a thin gray bar). There are two GenBank entries that align only to the reverse primer. One alignment of the primer pair to the GenBank entry L78833.1 is shown in Fig. 2. The forward primer aligns to the sequence L78833.1 on the forward strand (as indicated by Strand Plus/Plus) at nucleotides 3252..3270. The reverse primer aligns to the reverse strand (as indicated by Strand Plus/Minus) at nucleotides 3475..3457. Thus, the two primers will amplify the sequence from nucleotides 3252..3475 of the entry L78833.1. Retrieve the entry L78833.1 in Entrez, by clicking on it. The annotation shows that the amplified region covers the Exon 1a and the upstream sequence of the BRCA1 gene. Refer to the Note 1 for the multiple hits. You may perform similar search against the human genome BLAST database.