Primer- Definition, Types, Primer Design Online Tools, Uses

A primer is a short strand of nucleic acid, either RNA or DNA, that provides a starting point for DNA synthesis . It is bound to the template DNA and has a free 3`-OH end that can be extended by DNA polymerase . Primers are needed for DNA replication in living organisms and for polymerase chain reaction (PCR) in the laboratory .

In living organisms, primers are short strands of RNA that are synthesized by an enzyme called primase, which is a type of RNA polymerase. The primer serves to prime and lay a foundation for DNA synthesis by providing a 3`-OH end for DNA polymerase to attach new DNA nucleotides to an existing strand of nucleotides. The primers are removed before DNA replication is complete, and the gaps in the sequence are filled in with DNA by DNA polymerases.

In the laboratory, scientists can design and synthesize DNA primers with specific sequences that bind to sequences in a single-stranded DNA molecule. These DNA primers are commonly used to perform PCR to copy pieces of DNA or for DNA sequencing. PCR is a technique that uses repeated cycles of heating and cooling to amplify a specific region of DNA using a pair of primers that flank the target sequence. DNA sequencing is a technique that determines the order of nucleotides in a DNA molecule using primers that initiate the synthesis of complementary strands.

Primers are essential tools for molecular biology and biotechnology, as they enable the manipulation and analysis of DNA. Primers can be used for various purposes, such as cloning, gene expression, mutation detection, genotyping, gene editing, and more. Primers can also be modified with labels, such as fluorescent dyes or biotin, to facilitate detection or purification of the amplified or sequenced DNA.

Primers must be designed carefully to ensure specificity, efficiency, and accuracy of the desired reaction. Some factors that affect primer design include length, melting temperature, GC content, secondary structure, complementarity, and cross-hybridization. Various online tools are available to assist with primer design, such as Primer3, PrimerQuest, PerlPrimer, Oligo Primer Analysis Software, GenScript Real-Time PCR Primer Design, and AutoPrime.

Primers are classified into two main types based on their nucleic acid composition: DNA primers and RNA primers. Both types of primers have different roles and characteristics in biological processes and molecular techniques.

DNA primers

DNA primers are short synthetic oligonucleotides that are composed of deoxyribonucleotides. They are used in various in vitro techniques that require DNA synthesis, such as PCR, DNA sequencing, site-directed mutagenesis, and cloning. DNA primers have several advantages over RNA primers, such as:

• They are more stable and less prone to degradation by nucleases or chemical agents.
• They can be easily synthesized and modified with different labels or tags.
• They do not require any enzymes to initiate DNA synthesis, unlike RNA primers that need primase.
• They can be designed to have specific sequences and properties, such as melting temperature, GC content, and specificity.

However, DNA primers also have some limitations, such as:

• They may form secondary structures or dimers with themselves or other primers, which can affect the efficiency and accuracy of DNA synthesis.
• They may bind to non-target regions of the template DNA, which can result in non-specific amplification or sequencing.
• They may be affected by the presence of inhibitors or contaminants in the reaction mixture, which can reduce their activity or fidelity.

RNA primers

RNA primers are short RNA molecules that are composed of ribonucleotides. They are used in vivo for DNA replication, where they provide a free 3` hydroxyl group for DNA polymerase to start adding nucleotides to the growing strand. RNA primers have some advantages over DNA primers, such as:

• They can be synthesized de novo by the enzyme primase, which recognizes specific sequences on the template DNA and adds complementary ribonucleotides.
• They can be removed by the enzyme RNase H or by the exonuclease activity of DNA polymerase after DNA synthesis is completed, which prevents interference with downstream processes.
• They can initiate DNA synthesis at multiple sites along the template DNA, which increases the speed and efficiency of replication.

However, RNA primers also have some drawbacks, such as:

• They are less stable and more susceptible to degradation by nucleases or chemical agents.
• They cannot be used for in vitro techniques that require high temperatures or harsh conditions, such as PCR or sequencing.

• They may introduce errors or mutations into the newly synthesized DNA strand, due to the lower fidelity of primase compared to DNA polymerase.

  Comparison between DNA and RNA primers

DNA and RNA primers are both short sequences of nucleotides that serve as starting points for DNA synthesis. However, they have different roles and properties depending on whether they are used in vivo (in living cells) or in vitro (in laboratory experiments).

DNA primers

• DNA primers are used in vitro for DNA amplification techniques such as PCR (polymerase chain reaction) and DNA sequencing.
• DNA primers are composed of deoxyribonucleotides, which have a hydrogen atom at the 2` position of the sugar ring.
• DNA primers are usually 18 to 24 nucleotides long and are designed to be complementary to the target DNA sequence.
• DNA primers are stable at high temperatures and can withstand multiple cycles of heating and cooling during PCR.
• DNA primers remain a part of the newly synthesized DNA strand and do not need to be removed after the reaction.

RNA primers

• RNA primers are used in vivo for DNA replication, which is the process of copying the genetic material of a cell before cell division.
• RNA primers are composed of ribonucleotides, which have a hydroxyl group at the 2` position of the sugar ring.
• RNA primers are usually 10 to 20 nucleotides long and are synthesized by an enzyme called primase, which adds nucleotides to a short RNA primer that is already bound to the template DNA strand.
• RNA primers are unstable at high temperatures and can be degraded by enzymes called RNases.
• RNA primers are removed after completion of replication by another enzyme called exonuclease, which replaces them with DNA nucleotides.

Essential factors for Primer designing

Primer designing is a crucial step in PCR, DNA sequencing and other molecular biology applications that require specific and efficient amplification of a target DNA sequence. There are several factors that need to be considered when designing primers, such as:

• Primer length: The optimal length of a primer depends on the application and the complexity of the template DNA. Generally, primers between 18-24 nucleotides are preferred, as they provide enough specificity and stability for annealing to the template. However, longer primers may be required for complex templates or low-copy targets, while shorter primers may be suitable for simple templates or high-copy targets.

• Melting temperature (Tm): The melting temperature of a primer is the temperature at which half of the primer molecules dissociate from the template DNA. It is influenced by the primer length, GC content, salt concentration and other factors. A good primer should have a Tm between 52°C-56°C, and the difference between the Tm of the forward and reverse primers should not exceed 2°C. This ensures that both primers anneal to the template at the same optimal annealing temperature (Ta), which is usually calculated as Ta = 0.3Tm(primer) + 0.7Tm(product) - 14.9.

• GC content and clamp: The GC content of a primer is the percentage of guanine and cytosine bases in its sequence. A primer should have a GC content between 40-60%, as this provides a balanced stability and specificity for binding to the template. Moreover, a primer should have a GC clamp, which is one or two GC bases at the 3` end of the primer. This enhances the binding efficiency and reduces the chances of mispriming.

• End stability: The end stability of a primer is the strength of binding between the last few bases at the 3` end of the primer and the template DNA. A primer should have a high end stability, as this determines the initiation of DNA synthesis by the polymerase enzyme. A high end stability can be achieved by avoiding AT-rich sequences or mismatches at the 3` end of the primer.

• Restriction enzyme (RE) cut sites: Some applications may require adding RE cut sites to the primers, such as cloning or mutagenesis. In this case, it is important to choose an appropriate RE that does not cut within the target sequence or create unwanted mutations. Moreover, it is advisable to add a few extra bases (leader sequence) to the 5` end of the primer, as this improves the efficiency and accuracy of RE digestion.

These are some of the essential factors for primer designing that can help achieve optimal results in PCR and other molecular biology techniques. However, there are other factors that may also affect primer performance, such as secondary structures, dimers, repeats, runs and cross-homology. Therefore, it is recommended to use online tools or software to design and validate primers before using them in experiments.

Caution for designing PCR Primers

PCR primers are short synthetic oligonucleotides that anneal to the template DNA and serve as starting points for DNA polymerase to extend and amplify the target region. However, designing optimal PCR primers is not a trivial task, as there are several factors that can affect the specificity, efficiency and accuracy of the PCR reaction. Therefore, some caution is needed when designing PCR primers to avoid potential problems and ensure reliable results. Some of the common pitfalls and solutions for designing PCR primers are:

• Primer specificity: The primers should be specific to the target region and not bind to any other regions of the template DNA or to each other. This can be achieved by checking the primer sequences against the template sequence and avoiding regions with high similarity or repeats. Additionally, the primers should have a balanced GC content (40-60%) and a GC clamp (one or two GC bases at the 3` end) to increase their specificity and stability. The melting temperature (Tm) of the primers should also be similar (within 2°C) and match the annealing temperature (Ta) of the PCR reaction.

• Primer dimers: Primer dimers are formed when two primers anneal to each other by complementary base pairing, either partially or fully. This can reduce the amount of available primers for the target region and interfere with the PCR amplification. Primer dimers can be avoided by minimizing the complementarity between the primers, especially at the 3` end, where the extension occurs. The primer sequences can be analyzed using online tools or software to identify and eliminate potential primer dimers.

• Hairpin loops: Hairpin loops are formed when a single primer folds back on itself and forms a loop structure by intramolecular base pairing. This can prevent the primer from binding to the template DNA and reduce the efficiency of the PCR reaction. Hairpin loops can be avoided by avoiding regions with high self-complementarity or palindromes in the primer sequences. The primer sequences can also be analyzed using online tools or software to identify and eliminate potential hairpin loops.

• Secondary structures: Secondary structures are formed when the template DNA folds into complex shapes by intramolecular base pairing, such as stem-loops or quadruplexes. This can hinder the access of the primers and the polymerase to the target region and affect the PCR amplification. Secondary structures can be avoided by choosing regions with low complexity and low GC content in the template DNA. The template sequence can also be analyzed using online tools or software to identify and avoid potential secondary structures.

• PCR product size: The size of the PCR product affects the efficiency and accuracy of the PCR reaction. Generally, smaller products (<1 kb) are easier to amplify than larger products (>5 kb), as they require less time, less enzyme and less template DNA. Larger products also have a higher risk of contamination, degradation and nonspecific amplification. Therefore, it is advisable to design primers that generate products within an optimal range (100-500 bp) for most applications.

By following these guidelines and using appropriate online tools or software, PCR primers can be designed with caution and confidence to ensure successful PCR experiments.

Best Primer Design Online Tools

There are many online tools available for designing primers for PCR, sequencing and other applications. These tools can help you to select optimal primers based on various criteria such as specificity, melting temperature, GC content, primer-dimer formation and more. Here are some of the most popular and widely used online primer design tools:

• NCBI`s Primer-BLAST: This is a comprehensive tool that combines the Primer3 algorithm with BLAST search to find specific primers for your target sequence. You can input an accession number, a FASTA sequence or upload a file as your PCR template. You can also specify the range, length, melting temperature and GC content of your primers, as well as the product size and number of primers to return. The tool will check the specificity of your primers against a selected database and organism, and report any potential off-target amplification or primer-dimer formation. You can also choose to allow splice variants or exclude predicted transcripts from your search. The tool will display the primer sequences, their properties, their locations on the template and their BLAST alignments.

• IDT OligoAnalyzer: This is a simple yet effective tool for determining physical properties of oligo sequences up to 255 bases. You can input a single primer or a pair of primers and get information such as melting temperature, GC content, molecular weight, extinction coefficient, hairpin and dimer formation and more. You can also adjust the salt concentration and oligo concentration to see how they affect the primer properties.

• Eurofins Genomics Primer Design Tools: These are free online tools that use the Prime+ software of the GCG Wisconsin Package to design PCR and sequencing primers. You can input a FASTA sequence or upload a file as your template and specify the target range or positions for your primers. The tools will consider various constraints on the primer and product properties such as length, melting temperature, GC content, repeats, stability and more. The tools will also avoid primers with extensive self-dimer and cross-dimer formations and secondary structures. The results are scored according to the best predicted performance criteria. You can also save your selected primers in your shopping cart.

• GenScript Real-time PCR (TaqMan) Primer Design: This is a tool specifically suited for TaqMan primer and probe creation. It is nice and simple to use with minimal primer parameters to set. You can customize the PCR amplicon size, primer and probe Tm and choose to have the primer/probe crossing an exon junction. The tool will generate a list of primer-probe sets that match your criteria and show their locations on the template sequence.

• BatchPrimer3: This is a great tool for performing a more high-throughput primer design. You can input multiple sequences in FASTA format or upload a file as your templates and specify the common primer parameters for all sequences. The tool will use Primer3 to design primers for each sequence individually and output a table of primer pairs with their properties and product sizes.

These are some of the best online tools for primer design that you can use for your project. However, you should always validate your primers experimentally before using them for your intended application

Primer design Protocol/ steps/Process

There are different methods and tools for designing primers for PCR and other applications. Here we will describe a general protocol for designing primers using Primer-BLAST, a web-based tool that combines Primer3 and BLAST to find specific primers.

1. Enter your PCR template sequence as an accession number, a gi number, or a FASTA sequence. You can also upload a FASTA file. Optionally, you can specify a range of the template sequence to use for primer design.
2. Enter your desired PCR product size range, the number of primers to return, and the primer melting temperature range. You can also enter your own forward and reverse primers if you have them.
3. Choose the primer pair specificity checking parameters. This will determine how Primer-BLAST will search for potential off-target amplification using BLAST. You can select the database, the organism, the Entrez query, and the primer specificity stringency criteria. You can also enable or disable the search for splice variants and exclude certain types of sequences from the search.
4. Click on the "Get Primers" button to start the primer design and specificity checking process. This may take a few minutes depending on the size and complexity of your template sequence and the parameters you selected.
5. Review the results page. You will see a summary of your input parameters, a graphic view of your template sequence with the primer locations and product size, and a table of primer pairs with their properties and specificity information. You can also download the results as a text file or an Excel file.
6. Select the primer pair that best suits your needs based on the criteria such as product size, primer melting temperature, GC content, self-complementarity, 3` stability, and specificity score. You can also click on each primer pair to see more details such as the primer sequences, the BLAST alignments, and the secondary structure predictions.
7. Order your primers from a reliable supplier and test them in your PCR experiment.

This is a basic protocol for designing primers using Primer-BLAST. You may need to adjust some parameters or use other methods or tools depending on your specific application and goals. For more information and tips on primer design, you can refer to these sources .

Validation of Primers and Probes in qPCR

Once the designing of qPCR primers and probes has been done using available tools, insilico validation is to be performed by BLAST (insilico validation) for the confirmation of targeted gene sequences specificity. The algorithm of BLAST carries out sequence- similarity search against several databases with a set of gapped alignments of links to full database records. The query coverage and the maximum identity should be 100%. However, the BLAST program reports a statistical significance, called “expectation value”(E – value) for each alignment which is an indicator for finding the match by chance. E – values ≤ 0.01 convey the homologous sequences. E- value measures for assessing potential biological relationships.

Despite the fact that Insilco tools provide valuable feedback, the specificity of the qPCR assay using the designed primers and probes has to be validated empirically with direct experimental evidence. The specificity for a qPCR product can be affected by the presence of non-specific amplification and can be checked by analyzing the melting curves, also called dissociation curves, generating those qPCR protocols based on ds DNA binding dyes including SYBR green, since they bind to primer-dimer and other reaction artifacts producing a fluorescent signal. The melting curves can be carried out in all reported software programs for performing qPCR after amplification.

A quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting out and sequencing the band for verification. Primer dimers will often show up as a smear or band around 30-50 bp. Alternatively, fluorescently labeled probes can be used for greater specificity in a qPCR assay. The most common are hydrolysis probes (see Real-Time PCR Primer and Probe Chemistries for more details).

Applications of Primer Design

Primer design is a crucial step for various molecular applications that involve PCR, sequencing, hybridization, and SNP detection. Depending on the purpose and scope of the experiment, different types of primers may be required, such as degenerate primers, multiplex primers, nested primers, allele-specific primers, and probe primers. Some of the common applications of primer design are:

• PCR amplification: PCR is a widely used technique to amplify a specific region of DNA from a template. PCR requires a pair of primers that are complementary to the target sequence and flank it on both ends. The primers anneal to the template and serve as initiation points for DNA polymerase to extend and synthesize new strands. PCR can be used for various purposes, such as cloning, genotyping, mutation detection, gene expression analysis, and forensic science .
• DNA sequencing: DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. DNA sequencing often requires PCR to generate sufficient amounts of DNA for analysis. The primers used for PCR can also be used for sequencing, or new primers can be designed to cover specific regions of interest. Sequencing primers are usually shorter than PCR primers and have a higher melting temperature .
• Probe hybridization: Probe hybridization is a technique that uses a labeled nucleic acid probe to detect a complementary target sequence in a sample. The probe can be labeled with a fluorescent dye, a radioactive isotope, or a biotin molecule that can be detected by various methods. Probe hybridization can be used for applications such as microarray analysis, in situ hybridization, Southern blotting, and Northern blotting .
• SNP detection: SNP detection is the identification of single nucleotide polymorphisms (SNPs), which are variations in a single base pair in a DNA sequence. SNPs can be used as markers for genetic diversity, disease susceptibility, drug response, and evolutionary relationships. SNP detection can be performed by various methods that rely on primer design, such as allele-specific PCR, TaqMan probes, molecular beacons, and high-resolution melting analysis .

Primer design is an essential skill for molecular biologists and bioinformaticians who work with nucleic acid-based techniques. Primer design software can facilitate the process by providing automated tools for sequence retrieval, primer selection, primer evaluation, and primer visualization. However, primer design software cannot replace experimental validation and optimization of the primers in the laboratory setting. Therefore, primer design should be done with careful consideration of the application, the template sequence, and the experimental conditions.

Limitations of Primer Design

Primer design is a crucial step for successful PCR, DNA sequencing, cloning, and other molecular biology applications. However, primer design also has some limitations that need to be considered and overcome. Some of the common limitations are:

• Primer specificity: Primers should be specific to the target sequence and avoid binding to non-target regions or forming secondary structures such as hairpins, dimers, or loops. Non-specific binding or secondary structures can reduce the efficiency, accuracy, and yield of PCR or DNA synthesis. To avoid these problems, primers should be designed with optimal length, GC content, melting temperature, and annealing temperature. Additionally, primers should be checked for possible cross-hybridization with other sequences in the genome or database using tools such as BLAST.
• Primer degeneracy: Primers can be designed with degenerate bases to account for sequence variation or ambiguity in the target region. Degenerate bases are represented by IUPAC codes that indicate multiple possible nucleotides at a given position. For example, R can stand for A or G, and Y can stand for C or T. However, using too many degenerate bases can increase the complexity and cost of primer synthesis and lower the specificity and efficiency of PCR or DNA synthesis. Therefore, degenerate primers should be used sparingly and only when necessary. The number of degenerate bases depends on the nature of the target sequence and the desired level of coverage .
• Primer availability: Primers need to be synthesized chemically by specialized companies or facilities. The synthesis process can take time and money, depending on the length, quantity, and quality of the primers. Moreover, primers need to be stored properly to prevent degradation or contamination. Primers should be stored at -20°C in a dark and dry place, and aliquoted to avoid repeated freeze-thaw cycles. Primers should also be labeled clearly and checked for their concentration and purity before use.

Primer designing is a crucial step in PCR, DNA sequencing and other molecular biology applications that require specific and efficient amplification of a target DNA sequence. There are several factors that need to be considered when designing primers, such as:

• Primer length: The optimal length of a primer depends on the application and the complexity of the template DNA. Generally, primers between 18-24 nucleotides are preferred, as they provide enough specificity and stability for annealing to the template. However, longer primers may be required for complex templates or low-copy targets, while shorter primers may be suitable for simple templates or high-copy targets.

• Melting temperature (Tm): The melting temperature of a primer is the temperature at which half of the primer molecules dissociate from the template DNA. It is influenced by the primer length, GC content, salt concentration and other factors. A good primer should have a Tm between 52°C-56°C, and the difference between the Tm of the forward and reverse primers should not exceed 2°C. This ensures that both primers anneal to the template at the same optimal annealing temperature (Ta), which is usually calculated as Ta = 0.3Tm(primer) + 0.7Tm(product) - 14.9.

• GC content and clamp: The GC content of a primer is the percentage of guanine and cytosine bases in its sequence. A primer should have a GC content between 40-60%, as this provides a balanced stability and specificity for binding to the template. Moreover, a primer should have a GC clamp, which is one or two GC bases at the 3` end of the primer. This enhances the binding efficiency and reduces the chances of mispriming.

• End stability: The end stability of a primer is the strength of binding between the last few bases at the 3` end of the primer and the template DNA. A primer should have a high end stability, as this determines the initiation of DNA synthesis by the polymerase enzyme. A high end stability can be achieved by avoiding AT-rich sequences or mismatches at the 3` end of the primer.

• Restriction enzyme (RE) cut sites: Some applications may require adding RE cut sites to the primers, such as cloning or mutagenesis. In this case, it is important to choose an appropriate RE that does not cut within the target sequence or create unwanted mutations. Moreover, it is advisable to add a few extra bases (leader sequence) to the 5` end of the primer, as this improves the efficiency and accuracy of RE digestion.

These are some of the essential factors for primer designing that can help achieve optimal results in PCR and other molecular biology techniques. However, there are other factors that may also affect primer performance, such as secondary structures, dimers, repeats, runs and cross-homology. Therefore, it is recommended to use online tools or software to design and validate primers before using them in experiments.

PCR primers are short synthetic oligonucleotides that anneal to the template DNA and serve as starting points for DNA polymerase to extend and amplify the target region. However, designing optimal PCR primers is not a trivial task, as there are several factors that can affect the specificity, efficiency and accuracy of the PCR reaction. Therefore, some caution is needed when designing PCR primers to avoid potential problems and ensure reliable results. Some of the common pitfalls and solutions for designing PCR primers are:

• Primer specificity: The primers should be specific to the target region and not bind to any other regions of the template DNA or to each other. This can be achieved by checking the primer sequences against the template sequence and avoiding regions with high similarity or repeats. Additionally, the primers should have a balanced GC content (40-60%) and a GC clamp (one or two GC bases at the 3` end) to increase their specificity and stability. The melting temperature (Tm) of the primers should also be similar (within 2°C) and match the annealing temperature (Ta) of the PCR reaction.

• Primer dimers: Primer dimers are formed when two primers anneal to each other by complementary base pairing, either partially or fully. This can reduce the amount of available primers for the target region and interfere with the PCR amplification. Primer dimers can be avoided by minimizing the complementarity between the primers, especially at the 3` end, where the extension occurs. The primer sequences can be analyzed using online tools or software to identify and eliminate potential primer dimers.

• Hairpin loops: Hairpin loops are formed when a single primer folds back on itself and forms a loop structure by intramolecular base pairing. This can prevent the primer from binding to the template DNA and reduce the efficiency of the PCR reaction. Hairpin loops can be avoided by avoiding regions with high self-complementarity or palindromes in the primer sequences. The primer sequences can also be analyzed using online tools or software to identify and eliminate potential hairpin loops.

• Secondary structures: Secondary structures are formed when the template DNA folds into complex shapes by intramolecular base pairing, such as stem-loops or quadruplexes. This can hinder the access of the primers and the polymerase to the target region and affect the PCR amplification. Secondary structures can be avoided by choosing regions with low complexity and low GC content in the template DNA. The template sequence can also be analyzed using online tools or software to identify and avoid potential secondary structures.

• PCR product size: The size of the PCR product affects the efficiency and accuracy of the PCR reaction. Generally, smaller products (<1 kb) are easier to amplify than larger products (>5 kb), as they require less time, less enzyme and less template DNA. Larger products also have a higher risk of contamination, degradation and nonspecific amplification. Therefore, it is advisable to design primers that generate products within an optimal range (100-500 bp) for most applications.

By following these guidelines and using appropriate online tools or software, PCR primers can be designed with caution and confidence to ensure successful PCR experiments.

There are many online tools available for designing primers for PCR, sequencing and other applications. These tools can help you to select optimal primers based on various criteria such as specificity, melting temperature, GC content, primer-dimer formation and more. Here are some of the most popular and widely used online primer design tools:

• NCBI`s Primer-BLAST: This is a comprehensive tool that combines the Primer3 algorithm with BLAST search to find specific primers for your target sequence. You can input an accession number, a FASTA sequence or upload a file as your PCR template. You can also specify the range, length, melting temperature and GC content of your primers, as well as the product size and number of primers to return. The tool will check the specificity of your primers against a selected database and organism, and report any potential off-target amplification or primer-dimer formation. You can also choose to allow splice variants or exclude predicted transcripts from your search. The tool will display the primer sequences, their properties, their locations on the template and their BLAST alignments.

• IDT OligoAnalyzer: This is a simple yet effective tool for determining physical properties of oligo sequences up to 255 bases. You can input a single primer or a pair of primers and get information such as melting temperature, GC content, molecular weight, extinction coefficient, hairpin and dimer formation and more. You can also adjust the salt concentration and oligo concentration to see how they affect the primer properties.

• Eurofins Genomics Primer Design Tools: These are free online tools that use the Prime+ software of the GCG Wisconsin Package to design PCR and sequencing primers. You can input a FASTA sequence or upload a file as your template and specify the target range or positions for your primers. The tools will consider various constraints on the primer and product properties such as length, melting temperature, GC content, repeats, stability and more. The tools will also avoid primers with extensive self-dimer and cross-dimer formations and secondary structures. The results are scored according to the best predicted performance criteria. You can also save your selected primers in your shopping cart.

• GenScript Real-time PCR (TaqMan) Primer Design: This is a tool specifically suited for TaqMan primer and probe creation. It is nice and simple to use with minimal primer parameters to set. You can customize the PCR amplicon size, primer and probe Tm and choose to have the primer/probe crossing an exon junction. The tool will generate a list of primer-probe sets that match your criteria and show their locations on the template sequence.

• BatchPrimer3: This is a great tool for performing a more high-throughput primer design. You can input multiple sequences in FASTA format or upload a file as your templates and specify the common primer parameters for all sequences. The tool will use Primer3 to design primers for each sequence individually and output a table of primer pairs with their properties and product sizes.

These are some of the best online tools for primer design that you can use for your project. However, you should always validate your primers experimentally before using them for your intended application

There are different methods and tools for designing primers for PCR and other applications. Here we will describe a general protocol for designing primers using Primer-BLAST, a web-based tool that combines Primer3 and BLAST to find specific primers.

1. Enter your PCR template sequence as an accession number, a gi number, or a FASTA sequence. You can also upload a FASTA file. Optionally, you can specify a range of the template sequence to use for primer design.
2. Enter your desired PCR product size range, the number of primers to return, and the primer melting temperature range. You can also enter your own forward and reverse primers if you have them.
3. Choose the primer pair specificity checking parameters. This will determine how Primer-BLAST will search for potential off-target amplification using BLAST. You can select the database, the organism, the Entrez query, and the primer specificity stringency criteria. You can also enable or disable the search for splice variants and exclude certain types of sequences from the search.
4. Click on the "Get Primers" button to start the primer design and specificity checking process. This may take a few minutes depending on the size and complexity of your template sequence and the parameters you selected.
5. Review the results page. You will see a summary of your input parameters, a graphic view of your template sequence with the primer locations and product size, and a table of primer pairs with their properties and specificity information. You can also download the results as a text file or an Excel file.
6. Select the primer pair that best suits your needs based on the criteria such as product size, primer melting temperature, GC content, self-complementarity, 3` stability, and specificity score. You can also click on each primer pair to see more details such as the primer sequences, the BLAST alignments, and the secondary structure predictions.
7. Order your primers from a reliable supplier and test them in your PCR experiment.

This is a basic protocol for designing primers using Primer-BLAST. You may need to adjust some parameters or use other methods or tools depending on your specific application and goals. For more information and tips on primer design, you can refer to these sources .

Once the designing of qPCR primers and probes has been done using available tools, insilico validation is to be performed by BLAST (insilico validation) for the confirmation of targeted gene sequences specificity. The algorithm of BLAST carries out sequence- similarity search against several databases with a set of gapped alignments of links to full database records. The query coverage and the maximum identity should be 100%. However, the BLAST program reports a statistical significance, called “expectation value”(E – value) for each alignment which is an indicator for finding the match by chance. E – values ≤ 0.01 convey the homologous sequences. E- value measures for assessing potential biological relationships.

Despite the fact that Insilco tools provide valuable feedback, the specificity of the qPCR assay using the designed primers and probes has to be validated empirically with direct experimental evidence. The specificity for a qPCR product can be affected by the presence of non-specific amplification and can be checked by analyzing the melting curves, also called dissociation curves, generating those qPCR protocols based on ds DNA binding dyes including SYBR green, since they bind to primer-dimer and other reaction artifacts producing a fluorescent signal. The melting curves can be carried out in all reported software programs for performing qPCR after amplification.

A quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting out and sequencing the band for verification. Primer dimers will often show up as a smear or band around 30-50 bp. Alternatively, fluorescently labeled probes can be used for greater specificity in a qPCR assay. The most common are hydrolysis probes (see Real-Time PCR Primer and Probe Chemistries for more details).

Primer design is a crucial step for various molecular applications that involve PCR, sequencing, hybridization, and SNP detection. Depending on the purpose and scope of the experiment, different types of primers may be required, such as degenerate primers, multiplex primers, nested primers, allele-specific primers, and probe primers. Some of the common applications of primer design are:

• PCR amplification: PCR is a widely used technique to amplify a specific region of DNA from a template. PCR requires a pair of primers that are complementary to the target sequence and flank it on both ends. The primers anneal to the template and serve as initiation points for DNA polymerase to extend and synthesize new strands. PCR can be used for various purposes, such as cloning, genotyping, mutation detection, gene expression analysis, and forensic science .
• DNA sequencing: DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. DNA sequencing often requires PCR to generate sufficient amounts of DNA for analysis. The primers used for PCR can also be used for sequencing, or new primers can be designed to cover specific regions of interest. Sequencing primers are usually shorter than PCR primers and have a higher melting temperature .
• Probe hybridization: Probe hybridization is a technique that uses a labeled nucleic acid probe to detect a complementary target sequence in a sample. The probe can be labeled with a fluorescent dye, a radioactive isotope, or a biotin molecule that can be detected by various methods. Probe hybridization can be used for applications such as microarray analysis, in situ hybridization, Southern blotting, and Northern blotting .
• SNP detection: SNP detection is the identification of single nucleotide polymorphisms (SNPs), which are variations in a single base pair in a DNA sequence. SNPs can be used as markers for genetic diversity, disease susceptibility, drug response, and evolutionary relationships. SNP detection can be performed by various methods that rely on primer design, such as allele-specific PCR, TaqMan probes, molecular beacons, and high-resolution melting analysis .

Primer design is an essential skill for molecular biologists and bioinformaticians who work with nucleic acid-based techniques. Primer design software can facilitate the process by providing automated tools for sequence retrieval, primer selection, primer evaluation, and primer visualization. However, primer design software cannot replace experimental validation and optimization of the primers in the laboratory setting. Therefore, primer design should be done with careful consideration of the application, the template sequence, and the experimental conditions.

Primer design is a crucial step for successful PCR, DNA sequencing, cloning, and other molecular biology applications. However, primer design also has some limitations that need to be considered and overcome. Some of the common limitations are:

• Primer specificity: Primers should be specific to the target sequence and avoid binding to non-target regions or forming secondary structures such as hairpins, dimers, or loops. Non-specific binding or secondary structures can reduce the efficiency, accuracy, and yield of PCR or DNA synthesis. To avoid these problems, primers should be designed with optimal length, GC content, melting temperature, and annealing temperature. Additionally, primers should be checked for possible cross-hybridization with other sequences in the genome or database using tools such as BLAST.
• Primer degeneracy: Primers can be designed with degenerate bases to account for sequence variation or ambiguity in the target region. Degenerate bases are represented by IUPAC codes that indicate multiple possible nucleotides at a given position. For example, R can stand for A or G, and Y can stand for C or T. However, using too many degenerate bases can increase the complexity and cost of primer synthesis and lower the specificity and efficiency of PCR or DNA synthesis. Therefore, degenerate primers should be used sparingly and only when necessary. The number of degenerate bases depends on the nature of the target sequence and the desired level of coverage .
• Primer availability: Primers need to be synthesized chemically by specialized companies or facilities. The synthesis process can take time and money, depending on the length, quantity, and quality of the primers. Moreover, primers need to be stored properly to prevent degradation or contamination. Primers should be stored at -20°C in a dark and dry place, and aliquoted to avoid repeated freeze-thaw cycles. Primers should also be labeled clearly and checked for their concentration and purity before use.

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