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We highly recommend using one of the many computer software programs available to design primers. This will help to accurately calculate the melting temperature (Tm) and help to identify potential secondary hybridization sites and secondary structure issues. The following must be taken into consideration when designing sequencing primers:

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

PCR primers have been traditionally designed by thermodynamic interaction with the desired templates1,2. Primers are designed to increase two respectively significant base sequence specificity and reasonable GC content indicators. The high specificity can prevent mispriming in regions other than the target region, and the GC content of a primer is a major factor in determining the annealing temperature (Tm). Maintaining the Tm value optimally affects the amplification efficiency of primers being used3. The reference scaffold for primers with high PCR success has been determined based on the result of trials of up to around 1990 thermodynamic calculations1. Indices such as discontinuity of the same base are also empirically determined. The Tm value, which explains the specificity of binding to the template and possibly with the primer dimer, among others, are evaluated to determine the appropriate primer pair on each template. With this, some of the proposed software has been designed. The most frequently used primer design software include PrimerSelect4, Primer Express (Applied Biosystems Primer Express Software Version 3.0 Getting Started Guide, 2004), Primer Premier ( ), OLIGO 75, Primer36, and OMP2. Of these primer design softwares, Primer3 software provides both a primer design on the Web and a local program (Primer3_core) that corresponds to a large amount of primer design, that becomes a standard for PCR primer design. In particular, Primer3 added some thermodynamic findings in 2007 and 20122,7. Its revision in 2012 provided an added knowledge about DNA duplex stability8 which incorporated an algorithm for designing primers on the target9. This enabled a primer design even in the boundary regions of exons.

Current primer design techniques allow the design of primers that amplify the subject sequence with high probability resultant of combining thermodynamic theory alongside the experience of many researchers. However, it has not been designed to make predictions about a nucleotide sequence that is said to be "not amplifying" a known template. In some previous cases where amplification was performed with an unexpected template in a PCR experiment, knowledge-feedback was unfortunately not documented. Earlier contributions on PCR primer designs have incorporated these into modifications of thermodynamic laws before being compacted to a primer design software2,5. To indicate the presence of a particular DNA or RNA sequence, there is always a need to predict that no PCR will occur at sequences that are not of interest or prime importance. In pathogen detection, PCR primers are selected based on several preliminary experiments to confirm that PCR can predictably occur only with a specifically targeted pathogen10,11,12,13. Since false positives pose a major problem in detecting many pathogens including COVID-19, it is important to develop a method for predicting false positives ( -19/false-negatives-how-accurate-are-pcr-tests-for-covid-19/). Hence, if specificity of a primer pair can be predicted from nucleotide sequences of primers and templates, hindrances including false-positives can be readily corrected resulting to an accelerated research process.

To express the relationships of these schemas as words, we decided to express the hairpin, primer dimer, primer-template bond, and primer-PCR product bond as words. The strength of the primer-template bond on the forward and reverse flanks greatly influences the establishment of the PCR reaction. For combinations that are not of the original primer-template, the binding position needs to be determined by PCR from the possible binding of multiple primer-templates. With this, we constructed the words for the learning RNN.

In this study, pseudo-words were created based on primer hairpins, dimers, primer-template homology, and primer-PCR product homology. Predicting the priming position is expected to be particularly important among pseudo-words. This is because PCR is established based on the elongation of DNA from the priming position (Fig. 1). When designing the optimum primer as in the conventional case, the binding position of the primer has a long complementary region and high stability as compared with other positions. However, when comparing the complementarity between the template and the primer sequence, which was not originally designed, it is necessary to determine the priming position from a large number of candidates having similar length and stability of the complementary strand. Also, the effect of priming position was conveyed by expressing the priming position in capital letters. The accuracy of this pumping position affects the accuracy of the overall prediction, whereas, in addition to the complementarity with the base sequence and template of the primer, it becomes an amplified sequence or set (reverse for forward, forward for reverse). Thus, its relationship with the priming position is also affected. Therefore, it is ideally desirable to learn and predict this priming position by artificial intelligence. However, since the basic data is not available in this study, the stability of the complementary strand is predicted by the nearest neighbor method. The priming position that maximizes stability was predicted with the set of priming positions. For the prediction of free energy by the nearest neighbor method, in addition to the values reported so far, values extrapolated from those values were set and used. Since some of these numbers are simple extrapolations from the reported numbers, their accuracy is not yet guaranteed, hence, future improvements are still needed.

Primer3 picks primers for PCR reactions, considering as criteria:o oligonucleotide melting temperature, size, GC content, and primer-dimer possibilities,o PCR product size,o positional constraints within the source (template) sequence, ando possibilities for ectopic priming (amplifying the wrong sequence)o many other constraints.All of these criteria are user-specifiable as constraints, andsome are specifiable as terms in an objective function thatcharacterizes an optimal primer pair.3. CITING PRIMER3We request but do not require that use of this software be cited inpublications as* Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG.Primer3--new capabilities and interfaces.Nucleic Acids Res. 2012 Aug 1;40(15):e115.The paper is available at and * Koressaar T and Remm M.Enhancements and modifications of primer design program Primer3.Bioinformatics 2007;23(10):1289-1291.The paper is available at If you use masker function, please cite:* Koressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R and Remm M.Primer3_masker: integrating masking of template sequence with primer design software.Bioinformatics 2018;34(11):1937-1938.The paper is available at Source code available at -org/primer3.

The development of Primer3 is promoted by a small group ofenthusiastic scientists mainly in their free time.They do not gainany financial profit with Primer3.There are two groups of Primer3 users: end users, who runPrimer3 to pick their primers and programmers, who use Primer3in their scripts or software packages. We encourage both to usePrimer3.If you are an end user, we request but do notrequire that use of this software be cited in publicationsas listed above under CITING PRIMER3.If you are a programmer, you will see that Primer3 is nowdistributed under the GNU General Public License, version 2 or(at your option) any later version of the License (GPL2).As we understand it, if you include parts of the Primer3 sourcecode in your source code or link to Primer3 binary libraries inyour executable, you have to release your software also underGPL2. If you only call Primer3 from your software and interpretits output, you can use any license you want for your software.If you modify Primer3 and then release your modified software,you have to release your modifications in source code underGPL2 as well.We chose GPL2 because we wanted Primer3 to evolve and for theimprovements to find their way back into the main distribution.If you are programming a new web interface which runs Primer3,please include in the about page of the tool the sentence" uses Primer3 version ...".Please consider releasing your software under GPL2 as well,especially if you do not want to maintain it in the future.There is no need to ask us for permission to include Primer3in your tools.

1. The tag PRIMER_THERMODYNAMIC_ALIGNMENT was replaced by two new tags: - PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT which governs hairpinand oligo-oligo interactions. Therefore, whenever it is set to 1(the default) thermodynamic alignments will be used for oligo-oligointeractions and hairpins. - PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT which governs theoligo-template interactions and, when set to 1 (default is 0), willcause Primer3 to use the thermodynamic alignment against templates.The reason for this change is to provide the option of using theold alignment for oligo-template interactions when the thermodynamicalignment is too slow or the template is too long (there is a hardlimit THAL_MAX_SEQ=10k on the length of sequences involved inthermodynamic alignments).2. We changed the following default values:2.1. Changing default oligo temperature calculationsPRIMER_TM_FORMULA=1 (was 0)PRIMER_SALT_CORRECTIONS=1 (was 0)2.2. Making thermodynamic secondary structure calculations for oligos the default:PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1 (was 0)2.3. The following need reasonable values to support the changes in 2.1and 2.2 above:PRIMER_SALT_DIVALENT=1.5 (was 0.0)PRIMER_DNTP_CONC=0.6 (was 0.0)2.4. To make old defaults easily accessible, we added a command lineargument, --default_version=1, which directs primer3_core to use theold defaults. --default_version=2 directs primer3_core to use the newdefaults. The default is --default_version=2.2.5. IMPORTANT: because PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1,PRIMER_THERMODYNAMIC_PARAMETERS_PATH must point to the right location.This tag specifies the path to the directory that contains all theparameter files used by the thermodynamic approach. In Linux, thereare two *default* locations that are tested if this tag is notdefined: ./primer3_config/ and /opt/primer3_config/. For Windows,there is only one default location: .\primer3_config\. If the theparameter files are not in one these locations, be sure to setPRIMER_THERMODYNAMIC_PARAMETERS_PATH.2.6. Changed default for PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0 (was 1)(0 is almost always the the behavior one wants.)2.7. To get the behavior of -default_version=1 when -default_version=2set the following:PRIMER_TM_FORMULA=0PRIMER_SALT_CORRECTIONS=0PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=0PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=0PRIMER_SALT_DIVALENT=0.0PRIMER_DNTP_CONC=0.0PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=12.8. To get the behavior of -default_version=2 when -default_version=1set the following:PRIMER_TM_FORMULA=1PRIMER_SALT_CORRECTIONS=1PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=0PRIMER_SALT_DIVALENT=1.5PRIMER_DNTP_CONC=0.6PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=03. We changed the NULL value for SEQUENCE_FORCE_{LEFT,RIGHT}_START_END to-1000000, and made it an error to select PRIMER_FIRST_BASE_INDEX 2b1af7f3a8