PCR is a commonly used method to amplify DNA of interest in many fields such as biomedical research, diagnostic testing and forensic testing. While the outcome of PCR can be influenced by many other conditions such as the template DNA preparation and reaction conditions, designing a good pair of primers is a critical factor. A general requirement is that primers should have similar melting temperatures (Tm) and a balanced G/C content, but should avoid self-complementarity and hair-pin structure. Additional requirements may also apply in certain cases. For example, to avoid unwanted amplification of genomic DNA in reverse transcription PCR (RT-PCR), it is recommended that a primer pair span an intron, or that one of the primers be located at an exon-exon junction. Another concern is the possible impact of SNPs in the primer regions. Since a SNP may act as a mismatch in some cases, one should consider picking primers outside of such regions.
One critical primer property is the target specificity. Ideally, a primer pair should only amplify the intended target, but not any unintended targets. This is especially important for real time quantitative PCR (qPCR) where in many cases the amount of PCR product is represented by the total intensity of fluorescence incorporated into amplified DNA and any amplification of unintended targets can affect the measurement [1
]. Since different parts of chromosomes or transcripts may share some nucleotide similarity due to either homologous regions or fortuitous matches, it is not uncommon that a primer pair intended for one target will also bind to another one, resulting in non-specific target amplifications.
A number of studies have investigated the effects of mismatches between targets and primers and have shown that a target can be amplified even if it has a few mismatches to the primers [2
]. In general, mismatches towards the 3’ end affect target amplification much more than mismatches towards the 5’ end. Although the results from these studies vary and the precise relationship between mismatches and amplification is difficult to establish, the consensus is that a two base mismatch at the 3’ end generally prevents amplification. A single base mismatch (even at the very 3’ end), as well as a few mismatches in the middle or toward the 5’ end, still allows amplification, though at a reduced efficiency for some cases. Given the variable effects of the mismatches and the likelihood that users may have different criteria based on their own experimental conditions, it is important that a software tool should offer the capability to detect up to a few mismatches over the entire primer range and the flexibility to change the specificity settings. In this regard, it is worth pointing out that the BLAST program [6
] is in fact not an ideal tool for this purpose, as it uses a local alignment algorithm and does not necessarily return complete match information between the primer and target, particularly when the match is not perfect toward the primer ends.
A number of public software tools have been developed to aid the primer design process. Notably, the widely used Primer3 program [7
] designs primers based on a variety of parameters. Since it does not perform target analysis, users typically need to test the primer specificity using additional tools. However, this process is time-consuming and sometimes even impractical if the primers have too many database matches (as with a BLAST search, for example).
Several software programs, such as In-Silico PCR [8
] and Reverse ePCR [9
], do not design primers but rather determine the amplification targets of user-supplied primer pairs. However, even with the help of these tools, finding specific primers can still be a difficult process, because users often need to go through many candidate primers manually. In addition, since these software tools mostly use an index-based strategy, which requires computationally intensive pre-processing of the search database, they are limited by the availability of databases and are usually not sensitive enough to detect targets that have a significant number of mismatches to primers yet are potentially amplifiable.
It is therefore desirable to combine various elements of primer design requirements into one process such that users can simply input the template and obtain the desired target-specific primers. There are several existing programs that have addressed some aspects of this issue. Autoprime [10
] designs primers spanning exon junctions or introns so that the primers only target mRNA. However, it does not address the primer specificity issue. QuantPrime [11
] is a specialized tool to design target-specific primers for detecting mRNA in real time PCR. Likewise, the PRIMEGENS Sequence Specific Primer Design tool [12
] can also be used for specific primer design for a limited number of organisms. However, neither of these tools guarantees an accurate count of nucleotide matches between primer and target due to the fact that they both use a local alignment algorithm (i.e., BLAST) alone for the similarity search and thus may miss part of an alignment between primer and target [6
]. Other limitations in these tools include low target detection sensitivity, limited specificity stringency options, no or limited support for designing primers based on exon/intron boundary requirements and limited coverage of organisms in search databases.
We have developed Primer-BLAST as a general purpose public tool that helps users design target-specific primers. Primer-BLAST offers flexibility to accommodate different primer design needs. Users can either design new primers or check the specificity of pre-existing primers. Notably, Primer-BLAST incorporates a global alignment mechanism and is designed to be very sensitive in detecting potential amplification targets. Finally, it has the capability to place primers based on exon/intron boundaries and SNP locations. We are not aware of any other general purpose public tool that has integrated similar functionality to design target-specific primers.