Fu L et al. (2023), Competitive PCR with dual fluorescent primers e...

XB-ART-59834

Cell Biosci 2023 May 11;131:83. doi: 10.1186/s13578-023-01042-2.

Show Gene links Show Anatomy links

Competitive PCR with dual fluorescent primers enhances the specificity and reproducibility of genotyping animals generated from genome editing.

Fu L , Ma E , Okada M , Shibata Y .

???displayArticle.abstract???
Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through NHEJ mechanism is an error-prone process that often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels present difficulty for genotyping, often requiring labor intensive sequencing to obtain reliable results. Here, we developed a single-tube competitive PCR assay with dual fluorescent primers that allowed simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organisms.

???displayArticle.pubmedLink??? 37170319
???displayArticle.pmcLink??? PMC10173569
???displayArticle.link??? Cell Biosci
???displayArticle.grants??? [+]

???attribute.lit??? ???displayArticles.show???

Fig. 1. Co-PCR with two genotype-specific fluorescent primers faithfully distinguishes the three genotypes of a genome-editing target gene. Primers labelled with fluorescent IR700 and IR800 dyes that were specific for the wild type and mutant alleles of indicated target gene, respectively, were mixed together with a third primer common to both wild type and mutant alleles in single tube for competitive PCR to genotype animals with mutation in HAL2 A and MBD3 B. The PCR products were denatured and resolved on 15% Urea-PAGE gels. Fluorescent bands were digitally visualized on a LI-COR Odyssey Clx Scanner with the IR700 signal recorded as green and IR800 signal as red. The fluorescent densities were adjusted in each individual channel to the condition where the green and red fluorescent densities on PCR products of heterozygous mutant targets (Het) were about equal, which generated a yellow band in the merged field. The wild type (Wt) and homozygous mutant (Hom) bands were red and green, respectively. Note that genotype-specific primers could be either reverse primers (Rm and Rwt in panel A) or forward primers (Fm and Fwt in panel B), and should target the same region with only a short stretch of sequences different at the 3’-end (in purple letters). The arrows point to the PCR products and the star * indicates the unincorporated primers

Fig. 2. Genotype-specific PCR primers compete for targets to increase amplification specificity. Fluorescently labeled genotype-specific forward primers for MBD3 were used to pair with a common reverse primer individually or in combination in PCR reactions to amplify the gene-editing target region of MBD3 in genomic DNA isolated from wild type (Wt), heterozygous mutant (Het), and homozygous mutant (Hom) animals, respectively. The PCR products were resolved on a gel and scanned to visualize the fluorescent signals as in Fig. 1. Note that the wild type-specific forward primer (Fwt) non-specifically amplified the target region in the homozygous mutant template to produce a strong band when paired alone with the common reverse primer (circled in lane 4). However, the band was absent when both wild type-specific forward primer and mutant-specific forward primer were present together in a single tube dual fluorescent PCR reaction (circled in lane 10), likely due to more effective competition of the mutant-specific forward primer to bind to the mutant templates for PCR amplification. Similarly, the weak non-specific amplification of mutant-specific primer on the wild type template (circled in lane 5) when wild type-specific forward primer was absent was also inhibited in the dual fluorescent PCR reaction when the wild type-specific forward primer was also present (circled in lane 8). Ctrl: control PCR of DNA template from heterozygous animals as reference to adjust green and red signals for visualization

References [+] :

Buchholz, More similar than you think: Frog metamorphosis as a model of human perinatal endocrinology. 2015, Pubmed, Xenbase