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18. Juni 2023

A Beginner's Guide to Primer Express Software Version 3.0: How to Design Primers and Probes for Real-Time PCR

Primer Express Software Version 3.0 Free Download: A Guide for Beginners

If you are interested in designing your own primers and probes for real-time PCR applications, you may want to check out Primer Express Software Version 3.0. This software allows you to create custom primers and probes using TaqMan and SYBR Green I dye chemistries for gene quantitation and allelic discrimination (SNP) assays. It is compatible with various real-time PCR systems from Thermo Fisher Scientific, such as StepOne, StepOnePlus, 7300, 7500, 7500 Fast, 7900HT, ViiA 7, and QuantStudio.

Primer Express Software Version 3.0 Free Download

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In this article, we will guide you through the process of downloading, installing, and using Primer Express Software Version 3.0 for primer design. We will also provide some tips and tricks to help you optimize your primer design and performance.

What is Primer Express Software?

Primer Express Software is a tool that enables you to design your own primers and probes for real-time PCR applications. PCR, or polymerase chain reaction, is a technique that amplifies a specific region of DNA by using a DNA polymerase enzyme and two primers that bind to the opposite strands of the template DNA. Real-time PCR, also known as quantitative PCR or qPCR, is a variation of PCR that allows you to measure the amount of amplified DNA in real time by using fluorescent dyes or probes that emit signals during the amplification process.

There are two main types of real-time PCR chemistries: TaqMan and SYBR Green I. TaqMan assays use a pair of primers and a probe that hybridizes to the target sequence between the primers. The probe has a fluorescent reporter dye at one end and a quencher dye at the other end. When the probe is intact, the quencher suppresses the fluorescence of the reporter. However, when the probe is cleaved by the DNA polymerase during the extension phase of PCR, the reporter dye is separated from the quencher and emits fluorescence that can be detected by the real-time PCR instrument. The amount of fluorescence is proportional to the amount of target DNA in the sample.

SYBR Green I assays use only a pair of primers that flank the target sequence. SYBR Green I is a fluorescent dye that binds to double-stranded DNA nonspecifically. When the target DNA is amplified during PCR, more SYBR Green I molecules bind to it and increase the fluorescence signal that can be measured by the real-time PCR instrument. The amount of fluorescence is also proportional to the amount of target DNA in the sample. However, SYBR Green I can also bind to any other double-stranded DNA in the reaction, such as primer-dimers or nonspecific products, which can interfere with the accuracy and specificity of the assay. Therefore, SYBR Green I assays require more careful primer design and validation than TaqMan assays.

Primer Express Software allows you to design both TaqMan and SYBR Green I assays for gene quantitation and allelic discrimination (SNP) applications. Gene quantitation is used to measure the expression level or copy number of a gene of interest in a sample. Allelic discrimination (SNP) is used to detect single nucleotide polymorphisms (SNPs) in a gene of interest and differentiate between different alleles or variants.

Primer Express Software has a user-friendly interface that guides you through the steps of primer and probe design. It also has a built-in database of commonly used primers and probes for human, mouse, and rat genes that you can use or modify for your own assays. You can also import your own sequences or access online databases such as NCBI or Ensembl to select your template sequences.

Primer Express Software Version 3.0 is the latest version of the software that was released in 2009. It has some new features and improvements over the previous versions, such as:

Support for Windows 7 and Vista operating systems

Support for multiplexing up to four targets in a single reaction

Support for designing primers and probes for custom arrays

Improved primer design algorithm that considers primer secondary structures and primer-dimer formation

Improved specificity check that allows you to compare your primers and probes against multiple databases and select the best ones

Improved graphical user interface that displays the primer and probe locations, melting temperatures, GC content, and other parameters

Improved report generation that allows you to export your primer and probe information in various formats

How to download and install Primer Express Software

If you want to try out Primer Express Software Version 3.0, you can download a free trial version from the Thermo Fisher Scientific website. The trial version is valid for 30 days and has all the features of the full version except for saving, printing, and exporting your primer and probe information. To download the trial version, you need to register on the website and provide some basic information about yourself and your research interests. You will then receive an email with a link to download the software.

If you want to purchase the full version of Primer Express Software Version 3.0, you can contact your local Thermo Fisher Scientific sales representative or distributor. The price of the software depends on the type of license you choose: single-user, multi-user, or network license. You will receive a CD-ROM with the software and a license key that you need to activate the software.

To install Primer Express Software Version 3.0 on your computer, you need to have the following system requirements:

Windows XP (SP2 or higher), Windows Vista, or Windows 7 operating system

Pentium III processor or higher

256 MB RAM or higher

100 MB free hard disk space or higher

CD-ROM drive

Internet connection (optional)

The installation process is simple and straightforward. You just need to follow these steps:

Insert the CD-ROM into your CD-ROM drive.

The installation wizard will start automatically. If not, you can manually run the setup.exe file from the CD-ROM.

Follow the instructions on the screen to select your language, accept the license agreement, choose your destination folder, and complete the installation.

When prompted, enter your license key to activate the software.

You can now launch Primer Express Software from your desktop or start menu.

How to use Primer Express Software for primer design

Once you have installed Primer Express Software on your computer, you can start designing your own primers and probes for real-time PCR applications. The software has a wizard-like interface that guides you through the steps of primer design. You can also access the help menu or the user manual for more detailed instructions and explanations. Here are the basic steps for primer design using Primer Express Software:

Basic steps for primer design

Selecting a template sequence

The first step is to select a template sequence for your primer design. A template sequence is the DNA sequence that contains your target region of interest. You can select a template sequence from different sources:

You can use one of the predefined sequences from the built-in database of human, mouse, and rat genes. You can search for a gene by name, symbol, accession number, or keyword.

You can import your own sequence from a file in FASTA format.

You can access online databases such as NCBI or Ensembl and download a sequence by entering its accession number or keyword.

After selecting a template sequence, you can edit it by using the sequence editor. You can trim, reverse, complement, or annotate your sequence as needed. You can also select a specific region of your sequence as your target region by highlighting it or entering its start and end positions.

Choosing a primer type and chemistry

The next step is to choose a primer type and chemistry for your primer design. A primer type is the type of assay you want to perform with your primers. A chemistry is the type of fluorescent dye or probe you want to use with your primers. You can choose from the following options:

Quantitation: This primer type is used to measure the expression level or copy number of a gene of interest in a sample. You can choose between TaqMan and SYBR Green I chemistries for quantitation assays.

Allelic Discrimination (SNP): This primer type is used to detect single nucleotide polymorphisms (SNPs) in a gene of interest and differentiate between different alleles or variants. You can only use TaqMan chemistry for allelic discrimination assays.

Custom Array: This primer type is used to design primers and probes for custom arrays that can be used with the QuantStudio 12K Flex system. You can choose between TaqMan and SYBR Green I chemistries for custom array assays.

After choosing a primer type and chemistry, you can also specify the number of targets you want to multiplex in a single reaction. Multiplexing is the process of amplifying and detecting more than one target in a single reaction by using different fluorescent dyes or probes for each target. Primer Express Software Version 3.0 supports multiplexing up to four targets for TaqMan assays and up to two targets for SYBR Green I assays.

Setting primer parameters and specificity options

The next step is to set the primer parameters and specificity options for your primer design. Primer parameters are the criteria that determine the quality and performance of your primers, such as length, melting temperature, GC content, and product size. Specificity options are the settings that control how Primer Express Software checks the specificity of your primers against potential off-target sequences, such as databases, mismatches, and homology regions.

You can use the default values for the primer parameters and specificity options, or you can adjust them according to your preferences and needs. You can also save your custom settings as a template for future use. Here are some examples of the primer parameters and specificity options you can set:

Parameter/Option

Description

Default Value

Primer Length

The number of nucleotides in each primer

Primer Tm

The melting temperature of each primer, which is the temperature at which half of the primer molecules dissociate from their complementary strands

60C

Primer GC%

The percentage of guanine and cytosine nucleotides in each primer, which affects the stability and specificity of the primer binding

50%

Product Size

The length of the amplified DNA product between the primers, which affects the efficiency and sensitivity of the PCR reaction

75-150 bp for TaqMan assays100-200 bp for SYBR Green I assays

Database Search

The option to search your primers against one or more databases to check their specificity and avoid cross-reactivity with other sequences

EnabledYou can select from human, mouse, rat, or custom databases

Mismatch Tolerance

The number of mismatches allowed between your primers and any off-target sequences in the database search

0 for TaqMan assays1 for SYBR Green I assays

Homology Region SizeThe minimum length of a continuous region of homology between your primers and any off-target sequences in the database search15 bp for TaqMan assays16 bp for SYBR Green I assaysRunning the primer design and viewing the resultsThe final step is to run the primer design and view the results. After setting all the parameters and options, you can click on the Design button to start the primer design process. Primer Express Software will use its algorithm to generate a list of possible primers and probes that match your criteria and specificity options. The primer design process may take a few minutes depending on the complexity of your template sequence and the number of targets you want to multiplex.

When the primer design is completed, you can view the results in the results window. The results window displays the following information for each primer and probe pair:

Target name and number

Primer and probe sequences

Primer and probe locations on the template sequence

Primer and probe melting temperatures

Primer and probe GC content

Product size

Database search results

Primer secondary structures and primer-dimer formation

Probe secondary structures and quenching efficiency

You can also view a graphical representation of the primer and probe locations, melting temperatures, GC content, and product size by clicking on the Graph button. You can zoom in or out, change the scale, or print the graph as needed.

You can select the best primer and probe pair for your assay by comparing the results and choosing the ones that meet your requirements and expectations. You can also modify or optimize your primer and probe sequences by using the sequence editor or the optimization wizard. You can also test different primer parameters and specificity options by using the back button or the edit button.

When you are satisfied with your primer and probe design, you can save, print, or export your results in various formats, such as text, Excel, or XML. You can also order your primers and probes from Thermo Fisher Scientific or other suppliers by using the order button or the export button.

Tips and tricks for optimal primer design

Primer design is a critical step for successful real-time PCR applications. Poorly designed primers can result in low efficiency, low specificity, low sensitivity, or high variability of your PCR results. Therefore, it is important to follow some best practices and tips to optimize your primer design and performance. Here are some tips and tricks that you can use with Primer Express Software or any other primer design software:

Avoiding primer-dimer formation and secondary structures

Primer-dimer formation is a phenomenon where two primers bind to each other instead of the template DNA, forming a short double-stranded product that can interfere with the PCR reaction. Primer-dimers can reduce the efficiency and specificity of your PCR reaction, as well as generate false-positive signals in SYBR Green I assays. Secondary structures are loops or hairpins that form within a single primer or probe molecule, reducing its ability to bind to the template DNA. Secondary structures can also reduce the efficiency and specificity of your PCR reaction, as well as affect the fluorescence emission of your probes.

To avoid primer-dimer formation and secondary structures, you should follow these guidelines:

Use Primer Express Software or other tools to check the primer-dimer formation and secondary structures of your primers and probes before ordering them.

Avoid primers that have complementary sequences at their 3' ends, as these are more likely to form primer-dimers.

Avoid primers that have long runs of identical nucleotides (e.g., AAAAA), as these can also promote primer-dimer formation.

Avoid primers that have high GC content (>60%), as these can form more stable secondary structures.

Avoid primers that have self-complementarity or internal repeats, as these can also form secondary structures.

Avoid probes that have G or C nucleotides at their 5' end, as these can quench the fluorescence of the reporter dye.

Avoid probes that have secondary structures that overlap with the reporter or quencher dyes, as these can affect their fluorescence emission.

Using mixed sites and inosine bases for degenerate primers

Degenerate primers are primers that contain more than one possible nucleotide at one or more positions, allowing them to bind to multiple variants of a target sequence. Degenerate primers are useful for amplifying sequences that have high variability or unknown mutations, such as viral genomes or SNPs. However, degenerate primers can also reduce the efficiency and specificity of your PCR reaction, as they can bind to nonspecific sequences or form more primer-dimers.

To use degenerate primers effectively, you should follow these guidelines:

Use Primer Express Software or other tools to design degenerate primers based on a consensus sequence or a set of aligned sequences.

Use mixed sites (e.g., R for A/G, Y for C/T) to represent deg enerate nucleotides at a given position. Avoid using N (any nucleotide) as it can increase the degeneracy and nonspecificity of your primers.

Use inosine bases (I) to replace degenerate nucleotides at the 3' end of your primers. Inosine is a modified base that can pair with any of the four natural bases (A, C, G, T) with similar efficiency and stability. Using inosine can reduce the degeneracy and improve the specificity of your primers.

Limit the number of degenerate positions in your primers to a minimum. Ideally, you should have no more than two or three degenerate positions per primer, and no more than 16-fold degeneracy per primer pair.

Validate your degenerate primers by PCR and sequencing to confirm their specificity and performance.

Adding restriction sites and clamps to primers

Restriction sites are short sequences that are recognized and cut by specific enzymes called restriction endonucleases. Clamps are short sequences that are added to the 5' end of primers to increase their stability and specificity. Adding restriction sites and clamps to primers can be useful for various purposes, such as cloning, sequencing, or labeling of PCR products. However, adding restriction sites and clamps to primers can also affect their design and performance, as they can alter the length, melting temperature, GC content, and specificity of the primers.

To add restriction sites and clamps to primers effectively, you should follow these guidelines:

Use Primer Express Software or other tools to add restriction sites and clamps to your primers. You can select from a list of common restriction enzymes and clamps, or enter your own custom sequences.

Choose restriction sites and clamps that do not interfere with the primer binding or the PCR reaction. Avoid restriction sites and clamps that contain primer-dimer or secondary structure motifs, or that are present in the template sequence or the background DNA.

Choose restriction sites and clamps that are compatible with your downstream applications. For example, if you want to clone your PCR product into a vector, choose a restriction site that matches the vector's cloning site. If you want to sequence your PCR product, choose a clamp that does not interfere with the sequencing primer.

Adjust the primer parameters and specificity options accordingly after adding restriction sites and clamps to your primers. For example, if you add a long clamp to your primer, you may need to decrease the primer length or increase the primer Tm to maintain the optimal product size and primer annealing.