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ddPCR™

Droplet Digital polymerase chain reaction (ddPCR™) measures absolute numbers of nucleic acid encapsulated in water-oil droplets. It is a power tool for high-precision quantification of target DNA sequences, including absolute quantification of cDNA or gDNA, copy number variation (CNV) analysis and variant allele frequency (VAF) analysis for mutations.

Services

Absolute Quantification

  • Highly quantified measurement of target DNA (# of copies/µl)
  • No need for standard curves
  • Applicable to a wide range of concentrations

CNV Analysis

  • Determine the copy number of deletion or duplication of a locus
  • High resolution to discriminate consecutive copy number states
  • Target and reference probe design

VAF Analysis

  • Detection and analysis of rare mutations or sequences at high sensitivity and precision
  • Biomarker detection within background of a highly abundant counterpart

 

Application

Example. Verification of VAF in CRISPR-edited cell lines using ddPCR

The HCT116 cell lines carrying BRAF V600E/+ and EGFR L858R/+ respectively were generated using CRISPR-Cas9; after isolation of single heterozygous clones from both cell lines and cell expansion, genomic DNA was extracted, and the variant allele frequency was verified with probes targeting both wild type (VIC) and mutant (FAM) alleles using ddPCR. The ratio of copy numbers of BRAF wt and V600E mutant was at ~1:1, meaning one out of two alleles carrying the mutation; while the ratio of the EGFR wt and L858R mutant was at ~2:1, indicating the heterozygous clone was probably contaminated with wt cells.

  

Please contact GeneCopoeia at 1-866-360-9531 or inquiry@genecopoeia.com for the cell line services.

 

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