Fig. 3

Enhanced enrichment of long amplicons via refined nested PCR and magnetic beads selection. (A) Outline of the experimental approach for optimizing magnetic beads selection. (B) Primer design targeting the generation of short and long PCR products (332 bp for wild-type alleles and approximately 5.8 kb for alleles with integrated F8 cassettes) through nested PCR. (C) Evaluation of F8 allele enrichment in the 1st PCR products using regular PCR and touchdown PCR, assessed by qPCR. Error bars indicate mean ± SEM, based on data from 6 mice. Paired two-sided Student’s t-tests were used for statistical analysis. (D) Fine-tuning of the magnetic beads ratio to optimize F8 allele enrichment. The assessment was conducted through qPCR and nanopore sequencing of the 2nd PCR products derived from magnetic beads size-selected 1st PCR products. Error bars represent mean ± SEM, based on data from 6 mice. Paired two-sided Student’s t-tests were used for statistical analysis. (E) Gel electrophoresis display of 2nd PCR products with and without magnetic beads size-selection applied to the 1st PCR products. (F) Quantitative assessment of the relative enrichment of the F8 allele in the 2nd PCR products following 0.4x magnetic beads size selection of 1st PCR products. The evaluation was performed using qPCR (n = 15 PCR reactions) and nanopore sequencing (n = 10 PCR reactions). Error bars indicate mean ± SEM. Paired two-sided Student’s t-tests were conducted for statistical analysis