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Getting started with a basic project in FastPCR software is as easy as opening a new or existing file and then using copy—paste or starting to type. Output results are easy to save as Excel worksheet.
The separated output of the primer design is a list of primers, a set of primer pair sequences with their theoretical PCR products, and, for multiplex PCR, the result of the calculation of multiple-PCR primers for given target sequences.
In addition, the output shows optimal annealing temperature for each primer pair, the size of PCR product, and complete information for each designed primer and for each multiplex PCR product set.
The template length is not limited. It is important that all target sequences are prepared in the same format. FastPCR allows files to be opened in several ways: the original file can be opened as read-only for editing with text editors; files can be opened to memory without using text editors, which allows larger file s , up to Mb, to be analyzed; files within a folder can be selected and the files opened during task execution without the use of text editor program.
Additionally, the program can open files within a selected folder in order to join all these files in a text editor. For example, this feature can be applied to convert all files from a selected folder into a single file of FASTA sequences. Alternatively this feature allows splitting FASTA sequences to indi- vidual files in a particular selected folder. When a sequence file is opened, FastPCR displays the informa- tion about the opened sequence and its format.
The information status bar shows the number of sequences, the total sequence length in nucleotides , the nucleotide composition, and the purine, pyrimidine, CG percentage, and the melting temperature.
Files can be saved in various formats including. FASTA format has the highest priority and is simple, because the raw sequence is merely preceded by a definition line. Standardizing on a set of formats enables programs to be written that can read results from many different programs. The file may begin with as many lines of comment or description as required. Following the first line are lines that start with the sequence name, which is separated from the aligned sequence residues by white space.
For PCR Design Generalities applications, primers are usually 18—35 bases in length and should be designed such that they have complete sequence identity to the desired target fragment to be amplified.
The software can dynamically optimize the best primer length for the entered parameters. Primer pairs are analyzed for cross-hybridization and specificity of both primers and, optionally, selected with similar melting temperatures. The default primer design selection criteria are shown in Table 2.
It is possible to use predesigned primers or probes or, alternatively, predesigned primers can act as references for the design of new primers. The program accepts a list of prede- signed oligonucleotide sequences and checks the compatibility of each primer with a newly designed primer or probe.
The Tm for short oligonucleo- tides with normal or degenerate mixed nucleotide combinations is calculated in the default setting using nearest neighbor thermodynamic parameters [12, 13]. The CG content of an oligonucleotide is the most important factor that influences the Tm value. The melting temperature for mixed bases is calculated by averaging nearest neighbor thermodynamic parameters—enthalpy and entropy values—at each mixed site; extinction coefficient is similarly predicted by averaging nearest neighbor values at mixed sites [2, 3].
The melting temperature for primer probe self- or cross-dimers and for in silico PCR experiments with oligonucleotides having mismatches to the target is calculated using values for the thermodynamic parameters for a nucleic acid duplex. The FastPCR allows the choice of other nearest neighbor ther- modynamic parameters or simple non-thermodynamic Tm calcula- tion formulae.
For non-thermodynamic Tm calculation, we suggest using simple formulae; the Wallace—Ikatura rule [17] is often used as a rule of thumb when primer Tm is to be estimated at the bench. The two equations above assume that the stabilizing effects of cations are the same on all base pairs. Linguistic complexity measurements are performed using the alphabet-capacity L-gram method [20, 21] along the whole sequence length and calculated as the sum of the observed range xi , from 1- to L-size words in the sequence, divided by the sum of the expected E value for this sequence length.
LC values of 80 and higher allow for the rapid choice of the best primer or probe sequences. We specify an abstract parameter called primer quality PQ that can help to estimate the efficiency of primers for PCR.
Self-complementarity, which gives rise to possible primer-dimer and hairpin structures, reduces the final value. The PQ tries to describe the likelihood of PCR success of each primer; this value varies from for the best to 0 for the worst primers. PQ values of 80 and higher allow for the rapid choice of the best PCR primer pair combinations.
No adverse effects, due to the modification of the reaction buffer, chosen ther- mostable polymerases, or variations in annealing temperature, have been observed on the reproducibility of PCR amplification using primers with high PQ.
The FastPCR tool eliminates intra- and inter-oligonucleotide reactions before generating a primer list and primer pair candidates. Stable primer-dimer formation is very effective at inhibiting PCR because the dimers formed are amplified efficiently and compete with the intended target.
The program provides for the detection of alternative hydrogen bonding during primer-dimer and in silico PCR primer binding site detection. Guanine is the most universal base, because it forms the strongest base pair and the strongest mismatches. However, cytosine is the most discriminating base, because it forms the strongest pair and the three weakest mis- matches [23, 28].
G-rich and C-rich nucleic acid sequences can fold into four- stranded DNA structures that contain stacks of G-quartets [19]. These quadruplexes can be formed by the intermolecular associa- tion of two or four DNA molecules, dimerization of sequences that contain two G bases, or by the intermolecular folding of a single strand containing four blocks of guanines. The software predicts the pres- ence of putative G- and C-quadruplexes in primer sequences.
The gap sequences Xn may have varying lengths, and a rela- tively stable quadruplex structure may still be formed with a loop more than seven bases long, but in general, increasing the length of the gap leads to a decrease in structure stability. The most important values for estimating the Ta are the primer quality, the Tm of the primers, and the length of PCR fragment. The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm Tmmin.
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.
Because only one primer is used in these PCRs, the ends of the products must be reverse complements and thus can form stem-loops. The techniques of inter-repeat amplified polymorphism, including inter-retrotransposon amplified polymorphism IRAP , retrotransposon-microsatellite amplified polymorphism REMAP , inter-MITE amplification [31, 32], and Alu-repeat polymorphism [33, 34], exploit highly abundant, dispersed repeats as markers.
However, primers complementary to repetitious DNA may pro- duce many nonspecific bands in single-primer amplification and compromise the performance of unique PCRs. Alternatively, one can create a small, local, specialized library of repeat sequences based on those in Repbase [35] or TREP [36] and use this for searches.
However, the presence of multiple primer bind- ing sites does not necessarily lead to alternative amplification prod- ucts because, for successful amplification, the priming sites for both primers must be both located close to each other, in correct orien- tation, and sufficiently match the primer sequences. By default, FastPCR performs a test for nonspecific binding by repeat masking and low-complexity region detection and masking for each given sequence.
PCR Primer and Probe Design and Oligonucleotide Assembly and Analysis Additionally the software allows this test to be performed against a reference sequence or sequences e. Primers that bind to more than one location on given sequences will be rejected.
Even though the test for non- specific primer binding 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 hybridiza- tion of a primer with the template is a prerequisite for priming by DNA polymerase. The secondary nonspecific primer binding test is based on repeat masking using a quick local alignment screen which allows one mismatch within a hash index of mers between the refer- ence and input sequences.
The program Selected Task will only perform the selected task. Once the executive task is completed, the result is shown in the Result report text editor e. Once the user selects any attribute, the option attribute value field shows the default attributes value, which can then be modified. Typically, it is not necessary to use these commands to manage typical PCR primer design and these are applied to advanced tasks. Whilst PCR primer design will be performed independently for different targets, multiplex PCR primer design can be performed simultaneously with multiple amplicons within a single sequence as well as for different sequences, i.
By default, the software designs primers within the entire sequence length. Optionally, users can specify, individually for each sequence, multiple locations for both forward and reverse primers with the commands: 1.
A] nnnnnn[C Multiplex PCR can be carried out simulta- neously within a single sequence with multiple tasks as well as for different sequences with multiple tasks or a combination of both. Only cytosine not followed by guanidine CpG methylation will be replaced by thymine: 5. The program recognizes that a given sequence in the screening library dataset from loading the dataset file is the same as the sequence for which it is designing primers; this allows sequence selection to be made even if the selection matches the screening sequence perfectly.
This allows the same dataset to be used for both primer design and screening without having to make a new screening database for each sequence. In other words, a dataset that contains sequences A, B, C, and D can be used both for choosing primers and for checking primer specificity.
These primers or probes will be checked for compatibility inter-primer-dimer formation with newly designed primers. The number of preexisting primers is not limited to one or two; it can be as many as the user needs. The program is able to generate either long oligonucleotides or PCR primers for the amplification of gene-specific DNA fragments of user-defined length.
FastPCR provides a flexible approach to designing primers for many applications and for both linear and circular sequences. It will check if either primers or probes have secondary binding sites in the input sequences that may give rise to additional PCR products. The selection of the optimal target region for the design of long oligonucleotides is performed in the same way as for PCR primers.
The user can vary the product size or design primer pairs for the whole sequence without specifying parameters by using default or preset parameters. Preset parameters are speci- fied for various situations: for example, for sequences with low CG content, for long-distance PCR, for degenerate sequences, or for manual input.
A list of the best primer candidates and all compat- ible primer pairs that are optimal for PCR is generated. Users can specify, individually for each sequence, multiple locations for both forward and reverse primers within each sequence, whilst PCR design will be performed independently for different targets. Primers for multiplex PCRs can be designed from a single or from multiple targets Fig.
The program generates primer pairs and probes from the input sequences and shows the optimal annealing temperature for each primer pair and the sizes of PCR products together with information for each designed primer.
For compatible primer pairs, the annealing temperature and PCR product size are also provided. The simultaneous amplification of many targets reduces the number of reactions that needs to be performed; multiplex PCR thus increases throughput efficiency. The design of multiplex PCR assays can be difficult because it involves extensive computational analyses of primer pairs for interactions.
To achieve uniform amplification of the targets, the primers must be designed to bind with equal efficiencies to their targets. PCR conditions may need to be adjusted, for example, by increasing or decreasing the annealing temperature so that all products are amplified equally efficiently. To achieve uniform amplification, most existing multiplex primer design packages use primer melting temperature. In practi- cal terms, the design of almost identical Tas and Tms is very impor- tant.
The melting temperatures of the PCR products are also important because these are related to annealing temperature val- ues.
The annealing temperature must be optimal in order to maximize the likelihood of amplifying the target genomic sequences whilst minimizing the risk of nonspecific amplification.
Further improvements can be achieved by selecting the optimal set of primers that maximize the range of common Tms. The speed of calculation depends on the numbers of target sequences and primer pairs involved.
An alternative way to design compatible multiplex PCR primer pairs is to use predesigned primers as references for the design of new primers. The user can select input options for the PCR prod- ucts such as the minimum product size differences between the amplicons.
Primer design conditions can be set individually for each given sequence or all primers can be designed using the same values; selected settings have higher priority for PCR primer or probe design than the general settings. The results include primers for individual sequences, compatible primers, product sizes, and annealing temperatures.
The user may choose those alternative compatible primer pair combinations that provide the desired product sizes.
Using the program, researchers can select predesigned primer pairs from a target for their desired types of PCRs by changing the filtering conditions as mentioned above. For example, a conventional multiplex PCR requires differently sized at least by 10 bp amplicons for a set of target genes, so the value for the minimum size difference between PCR products can be selected.
In addition to avoid amplifying different amplicons of the same size, multiplex PCR must also minimize the generation of primer- dimers and secondary products, which becomes more difficult with increasing numbers of primers in a reaction.
To avoid the problem of nonspecific amplification, FastPCR selects primer pairs that give the most likelihood of producing only the amplicons of the target sequences by choosing sequences which avoid repeats or other motifs. The program allows the user to design not only compatible pairs of primers but also compatible single primers for different targets or sequences.
The input data can be either a single sequence with a minimum two internal tasks or many sequences with or without internal tasks. Most of the parameters on the interface are self-explanatory.
Optionally, the user is asked to provide the sequence and select oligonucleotide designing parameters. After specifying inputs and primer design options, the user can execute the primer design task. Once the design of the primer set is completed, the result will appear in two Result text editors: PCR primer design result and Multiplex PCR compat- ible pair primers.
Figure 8 shows the access to the PCR primer design output. The result text editor PCR primer design result will display the individual PCR primer design data, including the primer list and the compatible primer pairs for all the sequences and their internal tasks. The second Multiplex PCR compatible pair primers text editor collects final search results that are pre- sented as a list of the sets of the compatible primer pairs for multi- plex PCR.
The overall strategy of designing group-specific PCR primers uses a hash index of mers to identify common regions in the target sequences, followed by standard PCR primer design for the current sequence, and then testing the complementarity of these primers to the other sequences. FastPCR performs either multiple sequence alignment or accepts alignment sequence input, giving it the flexibility to use a different strategy for primer design.
If required, it can design degenerate PCR primers to amplify the polymorphic region of all related sequences. The FastPCR package designs large sets of universal primer pairs for each given sequence, identifies conserved regions, and generates suitable primers for all given targets.
The steps of the algorithm are performed automatically and the user can influence the settings for the primer design options.
The quality of primer design is dependent on sequence relationships, genetic similarity, and suitability of the consensus sequence for the design of good primers. The software is able to generate group-specific primers for each set of sequences independently, which are suitable for all sequences.
Once the primer set design is complete, the result will appear in the Result text editor, as the PCR primer design result. Figure 6 shows the access to the PCR primer design output Table 3. The result text editor PCR primer design result dis- plays the individual group-specific PCR primer design data, includ- ing the primer list and compatible primer pairs for all the sequences and their internal tasks where suitable primers are found.
In the case where an alignment has been input, the result text editor dis- plays only one group-specific PCR primer design set, including degenerate primers, in the primer list as well compatible primer pairs for all the sequences.
Microsatellites are ubiquitously distributed throughout Locus Search and PCR eukaryotic genomes, often highly polymorphic in length, and Primer Design thereby an important class of markers for population genetic studies. This method allows the detection of perfect and imperfect SSRs with a single, up to base, repeat motif.
Each entry sequence is processed for identification of SSRs and the SSR flanking regions are used to design compatible forward and reverse primers for their amplification by PCR. The default PCR primer design parameters are that the primers must be within bases from either side of the identified SSR. Often the sequences available around SSR loci are not suitable for designing good primers; the user can increase or decrease the distance from either side to find more efficient and compatible primer pairs.
The capabilities of FastPCR make it a complete bioinformatics tool for the use of microsatellites as markers, from discovery through to primer design. These oligonucleotides Synthesis should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand. Second, a given oligonucleotide sequence should be unique to avoid multiple nonspecific binding that may lead to incorrect assembly.
The software must dynamically choose the length of the oligonucleotides to ensure both the specificity and the uniform Tm. All oligonucleotides are designed without gaps between them. Other complementary regions are less important for assembling multiple fragments by PCR and ligation. The input data can comprise either a single or many sequences.
The user is asked to provide the sequence and select oligonucleotide design parameters. The interface allows changing Tm calculation parameters. The search process runs after pressing F5 or from menu bar or toolbox. The research result is presented as a list of oligonucle- otides for both strands. On each strand, all oligonucleotides are adjacent with no gap between neighboring primers. An oligonu- cleotide will overlap two oligonucleotides from the complemen- tary strand.
The algorithm pays attention to avoid nonspecific oligonucleotide hybridization to repeated regions. Protocol DOI: The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties.
The software provides comprehensive tools for designing primers for most PCR and perspective applications,. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction XCR , group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification Loop-mediated Isothermal AmplificationLoop-mediated isothermal amplification LAMP.
A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region s of interest for targeted next-generation sequencing, competitive allele-specific PCR KASP -based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features.
The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis.
FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. Loop-mediated isothermal amplification. Coronavirus Resources. Authors: Ruslan Kalendar 1 , 2. Ruslan Kalendar 1 , 2. Access enabled via: An Institution. PDF Full text Related articles. The software provides comprehensive tools for designing primers for most PCR and perspective applications, … more.
Citations 5. Recent citations: Yana Sboeva et al. Related articles Based on techniques. Butler , , Springer Protocols. Int J Mol Med 46 5 — Nucleosides Nucleotides Nucleic Acids 27 3 — Nucleic Acids Res 36 10 :e BMC Bioinformatics Bioinformatics 36 — Microbiol Immunol 63 10 — Anal Biochem — Bioanalysis 3 2 — Nat Protoc 3 5 —
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