Genetic screening using droplet digital PCR (ddPCR) was performed following a previously published protocol36 (link). Briefly, ddPCR was carried out using the Bio-Rad QX200 Droplet Reader, Droplet Generator, and QuantaSoft software (Bio-Rad). The reference assay for the two-copy, autosomal gene RPP30 was purchased from Bio-Rad (assay ID dHsaCP1000485, cat. no. 10031243). The hydrolysis probe–based PCR amplifications targeted to GFP (insert) and AMP (backbone) are as follows: GFP, primers (5′-GCCGACAAGCAGAAGAACG-3′, 5′-GGGTGTTCTGCTGGTAGTGG-3′) and hydrolysis probe (/56-FAM/AGATCCGCC/ZEN/ACAACATCGAGG/3IABkFQ/); AMP, primers (5′-TTTCCGTGTCGCCCTTATTCC-3′, 5′-ATGTAACCCACTCGTGCACCC-3′) and hydrolysis probe (/5HEX/TGGGTGAGC/ZEN/ AAAAACAGGAAGGC/3IABkFQ/). The GFP assay was run in duplex with the AMP assay as well as the genomic reference RPP30-HEX. The ratios of (GFP copies/μl)/(RPP30 copies/μl) were plotted against (AMP copies/μl)/(RPP30 copies/μl) to identify cohorts of clones for ongoing analysis.
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Droplet digital PCR for Genetic Screening
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Variable analysis
independent variables
- Genetic screening using droplet digital PCR (ddPCR)
- Ratios of (GFP copies/μl)/(RPP30 copies/μl)
- Ratios of (AMP copies/μl)/(RPP30 copies/μl)
- Bio-Rad QX200 Droplet Reader
- Droplet Generator
- QuantaSoft software (Bio-Rad)
- Reference assay for the two-copy, autosomal gene RPP30 (assay ID dHsaCP1000485, cat. no. 10031243)
- Hydrolysis probe–based PCR amplifications targeted to GFP (insert) and AMP (backbone)
- Positive control: The reference assay for the two-copy, autosomal gene RPP30 (assay ID dHsaCP1000485, cat. no. 10031243)
- Negative control: Not explicitly mentioned
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