PrimerDigital

The secondary (non-specific) binding test (Ctrl-K):



the program will analyse (using quick alignment) and look for the presence of possible additional complement sites for each primer (probe), which would result in non-specific PCR product.

The specificity of oligonucleotides is one of most important points; the optimal primer should hybridize only with the concrete target sequence. Particularly when genomic DNA is used as the template, it is possible that primers (probe) may complement to inverted repeated regions of the genome.
For human and animals genomes, where mostly spread short interspersed repetitive element (SINE) like Alu-repeats, primer sequences complement to it will produces or ether non-specific single-primer amplified PCR products or Alu-PCR for polymorphism detection.
For plants genomes, highly copied short or long direct repeats (LTRs) can become the result on non-specific amplification or inter-repeat amplification polymorphism (IRAP, REMAP or inter MITE amplification and else).

Usually, the site-specificity of the primer can be checked by performing a sequence homology search (e.g. blastn) through all known template sequences in the public genome database such as National Center for Biotechnology Information (NCBI), but it is no necessity to scan primers though NCBI databases to prove specificity of PCR amplification (any primers have many potential non-specific binding sites, but they not efficient for an exponential PCR amplification).
Our experience, the almost all problems with the secondary (non-specific) primer binding can be simply solving using any the BAC sequence(s) (about 100,000 bases) from analysing genome.

Other way is creation a small local specialized library of tandem and retrotransposons (especially SINE elements for animals and human genomes) repeats sequences. Repeat library created (use) from Repbase databases [Jurka et al, 2005].

By default, FastPCR non-specific binding test performed inside each given sequence. Additionally software allow to do this test throw reference sequence or sequences (BAC, YAC sequences or any long sequences or own database). Primers (probe) shown more then single location on current sequence or at least single location throw reference sequence will reject.

The non-specific primer binding test performed as default test for all primers and fully controlled by user and can be cancelled, select off: “The secondary (non-specific) binding test”. The secondary non-specific primer binding test based on the quick non-gaped local alignment screening primer sequence throws the reference and current sequence. Optionally, you can synchronize the secondary non-specific primer binding test with dataset by sequences names. Program recognize that a given sequence in the screening library dataset (from loading dataset file) is the same name as the sequence for which it is designing primer and allow primers to be made even though they match that screening sequence perfectly. This would allow the same dataset to be used for both primer design and screening against without having to make a new screening database for each sequence. In other word, for a dataset that contains sequences >A, >B, >C and >D; it will use the same dataset for choosing primers and for checking primer specificity. Due to the interest in multiplex polymorphism detection based on repeated DNA, it is important to support the ability to target genomic regions rich in repetitive elements. Program does not mask repeat regions on the target and reference sequences before searching for primer (probe) binging sites. It is possible design a primer (probe) to be specific to repetitive region. Single primer amplification for primer from inverted repeats is one most efficient PCR methods detection polymorphism.

Imitation unique PCR with checked “Excluding Synchronizing with dataset by Sequence Names”: load the same file in FASTA format to general editor and to “Load dataset file with reference DNA sequence(s)” and run the standard PCR.