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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December 20, 2023
Introduction # We have a list of genes, like:
EGFR MET TP53 And we want the genome positions plus 10 bases at each end for each exons, the results should like:
chr7 116672185 116672587 NM_000245_exon_0_10_chr7_116672196_f 0 + chr7 116699060 116700294 NM_000245_exon_1_10_chr7_116699071_f 0 + chr7 116731657 116731869 NM_000245_exon_2_10_chr7_116731668_f 0 + chr7 116739939 116740094 NM_000245_exon_3_10_chr7_116739950_f 0 + chr7 116740841 116741035 NM_000245_exon_4_10_chr7_116740852_f 0 + chr7 116755344 116755525 NM_000245_exon_5_10_chr7_116755355_f 0 + chr7 116757426 116757549 NM_000245_exon_6_10_chr7_116757437_f 0 + chr7 116757627 116757784 NM_000245_exon_7_10_chr7_116757638_f 0 + chr7 116758448 116758630 NM_000245_exon_8_10_chr7_116758459_f 0 + chr7 116759380 116759500 NM_000245_exon_9_10_chr7_116759391_f 0 + Method # UCSC Table Browser do this job perfectly, thanks.
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February 27, 2021
Introduction # We have a list of SNP rs numbers, like:
rs1002315756 rs1003815568 rs1004109382 rs1004635980 rs1008829651 And we want the genome positions for each SNPS, the results should like:
#chrom chromStart chromEnd name ref altCount alts shiftBases freqSourceCount minorAlleleFreq majorAllele minorAllele maxFuncImpact class ucscNotes _dataOffset _dataLen chr1 51453 51454 rs1004109382 C 1 T, 0 0 0 snv rareSome,rareAll, 409400514 36 chr1 51594 51599 rs1004635980 GGGGG 1 GGGG, 4 12 -inf,-inf,0.
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February 16, 2021
Introduction # MPprimer: a program for reliable multiplex PCR primer design
BACKGROUND: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered.
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