Ming, how do I design primers for qPCR?

Primer design is a crucial step for successful qPCR experiments. The National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/) is a great tool for this purpose.

 

Primer Design Principles
 

Primer length is typically 18-25 bp, with a GC content between 40-60%. The GC content of forward and reverse primers should be as close as possible. The primer Tm should be 55-65°C, with a difference of no more than 5°C (ideally less than 3°C) between forward and reverse primers.
 

1. Specificity
 

● Sequence Specificity: Primer sequences must be highly complementary to the target gene. Use tools like BLAST to verify specificity against the mouse genome database, ensuring primers only match the intended target.

● 3'-End Specificity: The 3' end should avoid multiple consecutive G or C bases, especially with more than 2 G/C in the last 5 bases. 'A' is generally best avoided at the 3' end; 'T' is preferred. A 3'-terminal 'A' can prime extension even with some mispairing.
 

2. Amplification Efficiency
 

● Product Length: Amplicon size should generally be controlled between 100-300 bp.

● Avoid Secondary Structures: Primers should avoid forming self-dimers, hairpin structures, or primer-dimers.
 

3. Primer Positioning
 

● Span Exon-Intron Boundaries: Design primers to anneal within exons, preferably spanning an exon-intron junction. This helps distinguish cDNA amplification from potential genomic DNA contamination.

● Avoid Repetitive or Hypervariable Regions: Steer clear of repetitive sequences and regions of high variability, which can lead to poor specificity or unstable amplification efficiency.

 

Step-by-Step Guide Using NCBI
 

1.  In the search bar, select the "Gene" database, enter the gene name, select the species (e.g., Mus musculus), and click "Search".

 

2.  Scroll down to find the "mRNA and Protein(s)" section. Click on the entry starting with "NM_" (RefSeq mRNA).
 

 

 

3.  Click "Pick Primers" (often under "Analyze this sequence" or similar). Set the product size range optimally to 80-150 bp (can be adjusted based on results). Set Tm to 60°C. Verify the correct species is selected. Click "Get Primers".
 

 

 

 

4.  Review Results. Multiple primer pairs with relevant information will be listed. Choose suitable primers based on the design principles above.
 

 

5.  Validate Specificity with BLAST. Return to the NCBI homepage, click "BLAST", and select "Primer-BLAST".

 

6.  Paste the selected forward (F) and reverse (R) primer sequences into the corresponding fields. Other parameters can usually be left as defaults. Click "Get Primers".
 

 

Interpret the Results: The output will show the primer pair's specificity against the selected genome, predicted amplicon details, and any potential off-target binding.
 

 

References:

[1] Relevant Zhihu article on primer design.

[2] Relevant WeChat article on primer design.

[3] National Center for Biotechnology Information (NCBI) website.

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