Chat now. Toggle navigation. Print Page. The following tips will improve these results: Concatenate the two primer sequences into one sequence separated by 5—10 Ns and enter into BLAST sequence box. Under Program Selection, select the Somewhat similar sequences blastn program. Under Algorithm parameters, decrease word size to 7, increase expect threshold to , and turn off the low complexity filter. Figure 1. A Only the best 3 alignments are shown blue lines at the bottom.
B Selecting one of them provides the actual sequence alignment. Sign up now. Significantly decreases noise, while increasing sensitivity and precision. Tubes or well plates. Probes available with different dye—quencher combinations in the same plate.
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 — 5 ]. 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. 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.
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.
We are not aware of any other general purpose public tool that has integrated similar functionality to design target-specific primers. The Primer-BLAST program consists of a module for generating candidate primer pairs and a module for checking the target specificity of the generated primer pairs. Primer3 is used to generate the candidate primer pairs for a given template sequence.
In order to increase the chance of finding specific primer pairs, at least one primer for a given primer pair should be located in regions where the PCR template does not share high similarity to unintended targets if possible. To achieve this, the PCR template sequence is submitted to MegaBLAST [ 14 ] for a fast search to identify regions that are highly similar to unintended sequences in the user-specified database. Primer3 is then instructed to place at least one primer for a given primer pair , if possible, outside of such regions.
The candidate primer pairs are then subject to the specificity checking process. Since Primer3 generates many candidate primer pairs and all of them may need to undergo specificity checking to obtain the specified number of target-specific primer pairs, the entire search process can be very long if each pair is searched with BLAST individually.
To solve this problem, we observe that any primer is essentially a sub-region of the PCR template and a single BLAST result using the template as a query should contain alignment information for all primer pairs. As a result, when a user supplies a template sequence to design new primers the template case , the template itself is submitted for a BLAST search just once, which greatly reduces the total search time.
To further minimize the search time, all regions on the template that do not contain candidate primers are masked out to avoid irrelevant BLAST hits. Since all candidate primer locations on the template are established by Primer3 already, amplification targets amplicons for all primer pairs can be identified using the single BLAST result above. A primer pair is deemed to be specific only if it has no amplicons on any targets other than the submitted template within the specificity checking threshold specified by the user.
Otherwise, it is considered non-specific. In addition to checking for amplicons between the forward and the reverse primers, Primer-BLAST also checks amplicons arising from either primer alone.
For example, the forward primer could also act as a reverse primer if it happens to match some regions on the minus strand of the template.
The default BLAST expect value cutoff is 30, for the primer-only case and it is typically adjusted much higher for the template case see below. This expect value is times higher than the standard BLAST program default the higher the expect value cutoff, the more sensitive the search and is necessary to ensure detection of targets that have a significant number of mismatches to primers yet are potentially amplifiable in PCR.
The expect value for a given BLAST match between a primer and a target is roughly proportional to the query sequence length given the same search database [ 6 ], but the query lengths used in the primer-only case and the template case are often very different. Therefore, there can be a large discrepancy in the expect values between the BLAST matches in the two cases, even though the same primer sequences are being aligned.
To resolve this issue, we internally adjust the specified expect value cutoff for the template case using the length of the artificial template from the primer-only case as a guide. This ensures that the BLAST results are equivalent between submitting a template and submitting primers only. Since a complete alignment between a primer and its targets is desired for accurate specificity checking, the NW global alignment algorithm [ 13 ] is incorporated into Primer-BLAST to realign any regions that are not fully aligned by BLAST.
Users can design new primer pairs by entering the DNA template alone or they can design one primer by entering the template plus the other pre-existing primer. If available, a RefSeq accession is recommended as it carries more information about the sequence [ 15 ], which allows Primer-BLAST to better identify the template and thus perform better primer specificity checking. The template length is limited to 50, bases.
Several database options are available for specificity checking with broad organism coverage. They are the databases of choice for designing new target-specific primers.
The traditional nr database, containing redundant entries, is also available and is mostly recommended for organisms that are not covered by other databases or for sequence entries not covered by the RefSeq databases.
In addition, users can specify the mismatch threshold above which any targets should be ignored i. It is not always possible to generate primers specific to a particular splice variant mRNA when the difference in exons is not sufficient to distinguish one from the rest. Therefore, Primer-BLAST offers the splice variant handling option that allows amplification of other variants from the same gene. The results page reports the specificity of the generated primers, a graphic summary of primer pairs in relation to the PCR template and certain features such as exons, as well as detailed information on each primer pair.
It will only show target-specific primers if found; otherwise, it will report all primers. In all cases, the actual targets will be listed along with detailed alignments between primers and targets. The specificity checking was performed against the NCBI RefSeq mRNA database with organism limited to human, since the goal was to find primer pairs that are specific to this transcript only among the human transcriptome.
This underscores the challenge if the same thorough examination of primer specificity task were to be performed manually. The average search time for designing new primers with default parameters using a human mRNA template of average length bases is 2. Numbers indicate the end positions of exons for variant 5. Note that several transcripts differ by 3 nucleotides due to use of slightly different splice sites even though they share same exons i. Example results of designing target-specific primers.
Note that while five primer pairs were returned as shown in graphic summary , due to space limitation, the figure shows details for the first primer pair only. Numbers in alignments indicate the start and end positions for primer and target. A dot. Not surprisingly, part or all of the forward primers picked by Primer-BLAST are located in exon 2 and all reverse primers are on the junctions between exon 3 and 5 since exon 4 is not present.
As an example, we obtained the primers for the same PCR template as above i. This primer pair indeed shows perfect matches to the ZNF gene transcript variant 5 as well as other transcript variants from the same gene and would generate a base amplicon. Interestingly, some other potential amplicons are also detected. One is an additional base amplicon present in the intended ZNF transcript. The other is the base amplicon from a different gene i. However, there are up to 5 mismatches between at least one of the primers and the targets, which is probably sufficient to prevent amplification interference or non-specific amplification.
Nevertheless, users can scrutinize this result and make judgment based on their own experimental experiences. Specificity checking of pre-existing primers. This search was performed by entering the forward and reverse primers without entering any template.
While the results indicated all 7 transcript variants from the ZNF gene have the same amplicons, this figure shows the details only for variants 1 and 5 due to space limitation. As shown above, Primer-BLAST is, by default, capable of detecting potential amplification targets that have up to 5 mismatches to a primer. Primer-Blast achieves this result by using highly sensitive BLAST parameters as well as an additional NW global alignment algorithm to ensure a complete alignment between the primer and its target.
However, one caveat is that the BLAST algorithm [ 6 ] requires a minimum stretch of nucleotide matches word size between the query and target and any tools using BLAST as search algorithm are subject to this limitation. Assuming a random distribution of mismatch locations, it is possible to calculate the number of possible arrangements of 18 matches and 2 mismatches. Ideally, a comparison of detection sensitivity would be to directly test the specificity checking modules across all tools using primers generated from a third party for example, primers generated by Primer3.
Unfortunately, this option is not available as Primer-BLAST is the only tool that offers specificity checking directly i.
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