July 21, 2025
Designing primers with excellent cross-species specificity using MP-Ref (demo link).
Select the target database; Enter the targets in BED format; Also select “dog” and “E. coli” as backgrounds databases; And set “Min=0 Max=0” both for “Number of Off-targets” and “Amplicon Count on Background Genomes”; Download the primers and open it in Excel; Copy the primers and paste to MFEprimer for validation: https://m4.igenetech.com/spec/. Select the three databases which we used for designed primers.
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July 21, 2025
What’s “Long Target Regions” # If next-generation sequencing (NGS) or short-read sequencing (e.g., PE150) is used to validate amplicons, target regions longer than 200 bp should be considered long target regions (LTRs). This is because the total amplicon length (including primer sequences) should not exceed 300 bp.
The Primer Design Strategy for “Long Target Regions” # For long-range genotyping (LRG) targets > 200 bp, a tiling primer design strategy is typically required, often necessitating amplification in two separate tubes.
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July 21, 2025
Primer Design for SMN1 Whole Region # The whole genome sequence of SMN1 has a near identical repeat sequences is from SMN2. Primers for SMN1 also can amplify sequences from SMN2 and can’t be avoidable. So for NGS validation, mutations from amplicons sequencing may need to further validation, like Sanger sequencing, which design primers with amplicon size large engouth to skip the repeat region.
https://m4.igenetech.com/muld/demo/e7cd1164-3441-4a50-8c76-1862ef09f6aa
Input: chr5 70924941 70953012; Genome: hg38; Set “Min=0” for “Minimum Nucleotide Differences”, otherwise, MP-Ref will run 20+ hours to find very specific primers and will fail eventually.
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July 21, 2025
This is an example for desinging primers with different amplicon sizes for gel electrophoresis (demo link).
Select the target database; Enter the targets in BED format; Choose “Validation method” = “[Multiplex] Size discrimination, e.g., gel electrophoresis”;. And set the “Product Size Min Difference” = 20, this parameter requies the size difference of each amplicons should >= 20 bp. As expected, the three amplicons have enough size differences which we can discriminate them by gel electrophoresis.
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July 21, 2025
Designing primers capable of amplifying both genome A and genome B simultaneously using MP-Ref (demo link)。
Select the target database: this time we design primers for mouse and requires these primers should also have exactly one amplicon in human genome. Please be noted that we also set “Min nonspecific amplicon Tm” = 57, a bigger higher Tm. This is to tell the server we need a good (with Tm close to the target amplicon in mouse genome) amplicon for human genome.
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July 21, 2025
This is an example of designing singleplex primers using MP-Ref (demo link).
Select the target database; Enter the targets in BED format; Choose “Validation method” = “[Singleplex] Sanger sequencing”;. Below are the results.
July 21, 2025
What’s the “STR” mode? # Short Tandem Repeats (STRs) are regions within the genome consisting of multiple repeating DNA sequences. The STR mode is specifically tailored for designing primers to amplify regions flanking STRs or any target regions intended to be amplified in their entirety.
In scenarios where the target region’s size exceeds the maximum allowed product size, a tiling design strategy is employed with a default setting of two tubes to ensure 100% coverage of the target.
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