The secondary non-specific binding test; alternative amplification

The specificity of the oligonucleotides is one of the most important factors for good PCR; optimal primers should hybridize only to the target sequence, particularly when complex genomic DNA is used as the template. Amplification problems can arise due to primers annealing to repetitious sequences (retrotransposons, DNA transposons, or tandem repeats). Alternative product amplification can also occur when primers are complementary to inverted repeats and produce multiple bands. This is unlikely when primers have been designed using specific DNA sequences (unique PCR). However, the generation of inverted repeat sequences is exploited in two common generic DNA fingerprinting methods - RAPD and AP-PCR. Because only one primer is used in these PCR reactions, the ends of the products must be reverse complements and thus can form stem-loops.

The techniques of inter-retrotransposon amplification polymorphism (IRAP), retrotransposon-microsatellite amplification polymorphisms (REMAP), inter-MITE amplification, and Alu-repeat polymorphism have exploited these highly abundant dispersed repeats as markers. However, primers complementary to repetitious DNA may produce many non-specific bands in single-primer amplification and compromise the performance of unique PCRs. A homology search of the primer sequence, for example using "blastn" against all sequences in GenBank or EMBL-Bank, will determine whether the primer is likely to interact with dispersed repeats. Alternatively, one can create a small local specialized library of repeat sequences based on those in Repbase or TREP.

The mismatches at the 3'end of the primers affect target amplification much more that mismatches at the 5'end. A two base mismatch at the 3'end of the primer is preventing amplification. A single base mismatch as well as several mismatches at the 5'end of the primer is allowed amplification, with reducing efficiency of the amplification.

On the other hand, the presence of multiple primer binding sites does not necessarily lead to an alternative amplification, because are necessary for the amplification both primers located close to each other. The close location of the primers at correct orientation to each other and efficient binding of each DNA target is determine the probability of alternative amplification.

By default, the FastPCR performs a non-specific binding test for each given sequence. Additionally the software allows this test to be performed against a reference sequence or sequences (e.g. BAC, YAC) or one's own database. Primers that bind to more than one location on current sequences will be rejected. Even though the non-specific primer binding test is performed as a default for all primers, the user may cancel the operation.

Identification of secondary binding sites including mismatched hybridization is normally performed by considering the similarity of the primer to targets along the entire primer sequence. An implicit assumption is that stable hybridization of a primer with the template is a prerequisite for priming by DNA polymerase. The FastPCR pays particular attention to the 3' end portion of the primer and calculates the similarity of 3'end of the primer to target (the length is chosen by the user) to determine the stability of the 3'-terminus. The secondary non-specific primer binding test is based on a quick, non-gapped local alignment (that allows one mismatch within a hash index of 9-mers) screening between the reference and input sequence.

The software offers flexible specificity stringency options. User can specify the number of mismatches that primers must have to unintended targets at 3'end region where these mismatches must be present. The default specificity settings are that at least one mismatch in the last five bases at the 3'end of primer.

Optionally, user 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:

pcr options