![]() PCR is a commonly used method to amplify DNA of interest in many fields such as biomedical research, diagnostic testing and forensic testing. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. First, the primers flanking regions of interest are generated either manually or using software tools then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. The process of designing specific primers typically involves two stages. Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. Not for Use in Diagnostic Procedures.Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.įor Research Use Only.If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences. Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences).Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains.Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. Try to make the melting temperature (Tm) of the primers between 65☌ and 75☌, and within 5☌ of each other.The shorter the primers are, the more efficiently they will bind or anneal to the target. Specificity usually is dependent on length and annealing temperature. A good length for PCR primers is generally around 18-30 bases.Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding.Here are some guidelines for designing your PCR primers: In order to achieve successful DNA amplification, it’s important to start off with the right primer. Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design.
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