Tips and New Tools for Primer Design

Good primer design is essential for a successful PCR. There are many factors to take into account when designing the optimal primers for your gene of interest, but even setting all of the necessary parameters that will promise a successful experiment, is not enough. It might be even more important to work with one platform to design primers, manage inventory and share all this data with your lab colleagues in order to make your work in the lab much more efficient.

To answer these specific needs, Genome Compiler has released its' new powerful feature – the Primer Library tool. It allows you to import primers from your computer or add primers from existing projects to Primer Libraries stored inside Genome Compiler's Materials Box. It is then possible to attach primers from any one of these libraries to any project sequence you work on and also share these libraries with others.

So, how does it work?

Working with this powerful tool is simple; a short explanation will help you get it started. All you need to do is to create a new Primer Library folder inside the Materials Box. The Primer Library opens as a new tab and contains the following information about primers in a tabular format:

Primer Name
Description
Annealing sequence
Overhang sequence
Length
% GC
Tm
Last date it was modified and by which user
An option for you to mark whether you have it "In Inventory" or not.

After creating your Primer Library, you can import primers to the Library from your computer and from existing projects, or create new primers directly from within the Primer Library table. The file formats supported for importing primers are:

Vector NTI primer database (.oa4)
Plasma DNA database (.pdat)
Comma Separated (Name,Sequence,Overhang)
Tab Separated (Name Sequence Overhang)
Semicolon Separated (Name;Sequence;Overhang)

What is it good for?

Storing your primers inside Genome Compiler Primer Libraries allows you to simultaneously search the libraries for primers which anneal to your opened project sequence. Hence you can avoid re-designing and ordering primers which you already have stored in your inventory. Using the “Attach” primer function, Genome Compiler will search your specified primer libraries for exact matches of entire primer annealing sequences in the opened project. You can also refine your search in the advanced settings that allow you to:

Search for a desired number of primer bases from the 3' end.
Set the amount of mismatches allowed between a sequence and the primers.
Specify a search region within the project sequence according to the 3' position of the primer.
Search within a specific primer Tm range.

Selecting "Find Primer" will open up a results list of all of the primers that match the project according to your settings – just "Attach" the desired primer/s to your project. You can edit your primers anytime via the project Primer Dialogs and also by accessing the Primer Library, but notice that in order to avoid a change in all of the projects the primer is attached to, duplicate the primer first and then make the desired edits.

Share it with your friends in the lab

Another reason to use Genome Compiler's Primer Library is the ability to share it and manage the primers' inventory together. Sharing the Primer Library is done by simply right clicking on the Primer Library folder you wish to share in the Materials Box and selecting "Share". Decide if you'd like to grant 'Read/Write' or 'Read only' access and then enter your colleague's email. You and your collaborators will then be able to work with one streamlined Library for all of your primers available in the lab. It will be possible to access and edit the primers inside the library any time, track the date and author of primer edits, attach primers to any project you are working on and check the primer availability.

Finally, let's conclude with 12 tips for a successful primer design:

The primer length should be between 18 to 25 nucleotides long.
The melting temperature (Tm) should range between 55°C and 65°C. If the T_m of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
The Tm difference between primers in a primer pair should not be greater than 5°C.
GC content: between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
In the last 5 bases at the 3' end of the primer, make sure that there are at least 2 G or C bases (GC clamp).
Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting.
Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
Verify your primers' specificity so they won't bind to other genomic regions through NCBI Primer BLAST or UCSC in-silico PCR.
In order to verify the Tm, try an annealing temperature gradient PCR reaction to find the optimal Tm according to your primer and enzyme.