Primer-BLAST is a new software tool designed to help users design target-specific primers for polymerase chain reaction (PCR). It combines BLAST with a global alignment algorithm to ensure full primer-target alignment and is sensitive enough to detect targets with significant mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step and check the specificity of pre-existing primers. It also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. PCR is a commonly used method to amplify DNA of interest in many fields such as biomedical research, diagnostic testing, and forensic testing. Designing a good pair of primers is a critical factor in PCR. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. It is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast. Primer-BLAST uses a global alignment algorithm to ensure accurate specificity checking. It is more sensitive than BLAST, which is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Primer-BLAST is run on a farm of machines at the NCBI to provide faster and more reliable service to users. It can handle input such as Genbank accessions, FASTA files, or primers from another source. It also takes advantage of rich information from NCBI sequence databases to support other requirements such as placing primers based on intron/exon boundaries and SNP locations. Primer-BLAST is a general-purpose tool that offers a level of sensitivity and usability not found in other tools. It offers flexible options to meet various specificity stringency requirements and uses a BLAST search (with a local alignment) to quickly identify as many targets as possible, but follows that up with a global alignment to get an accurate count of mismatches over the entire primer if BLAST alignments only cover part of the primer length. It is freely available for all users and the source code for the blast search and primer specificity checking are in public domain.Primer-BLAST is a new software tool designed to help users design target-specific primers for polymerase chain reaction (PCR). It combines BLAST with a global alignment algorithm to ensure full primer-target alignment and is sensitive enough to detect targets with significant mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step and check the specificity of pre-existing primers. It also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. PCR is a commonly used method to amplify DNA of interest in many fields such as biomedical research, diagnostic testing, and forensic testing. Designing a good pair of primers is a critical factor in PCR. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. It is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast. Primer-BLAST uses a global alignment algorithm to ensure accurate specificity checking. It is more sensitive than BLAST, which is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Primer-BLAST is run on a farm of machines at the NCBI to provide faster and more reliable service to users. It can handle input such as Genbank accessions, FASTA files, or primers from another source. It also takes advantage of rich information from NCBI sequence databases to support other requirements such as placing primers based on intron/exon boundaries and SNP locations. Primer-BLAST is a general-purpose tool that offers a level of sensitivity and usability not found in other tools. It offers flexible options to meet various specificity stringency requirements and uses a BLAST search (with a local alignment) to quickly identify as many targets as possible, but follows that up with a global alignment to get an accurate count of mismatches over the entire primer if BLAST alignments only cover part of the primer length. It is freely available for all users and the source code for the blast search and primer specificity checking are in public domain.