Droplet digital PCR and mile‐post analysis for the detection of F8 int1h inversions

Abstract

Background F8 int1h inversions (Inv1) are detected in 1%–2% of severe hemophilia A (HA) patients. Long‐range polymerase chain reaction (PCR) and inverse‐shifting PCR have been used to diagnose these inversions. Objectives To design and validate a sensitive and robust assay for detection of F8 Inv1 inversions. Methods Archival DNA samples were investigated using mile‐post assays and droplet digital PCR. Results Milepost assays for Inv1 showing high specificities and sensitivities were designed and optimized. Analysis of four patients, two carrier mothers, and 40 healthy controls showed concordance with known mutation status with one exception. One patient had a duplication involving exons 2–22 of the F8 gene instead of an Inv1 mutation. DNA mixtures with different proportions of wild‐type and Inv1 DNA correlated well with the observed relative linkage for both wild type and Inv1 assays and estimated the limit of detection of these assays to 2% of the rare chromosome. Conclusions The milepost strategy has several inherent control systems. The absolute counting of target molecules by both assays enables determination of template quantity, detection of copy number variants, and rare variants occurring in primer and probe annealing sites and estimation of DNA integrity through the observed linkage. The presented Inv1 milepost analysis offers sensitive and robust detection and quantification of the F8 int1h inversions and other rearrangements involving intron 1 in patients and their mothers.

Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

“…The same non-ITR assays used to characterize the encapsidated viral vector genome can be incorporated into a higher level viral vector genome integrity analysis with a milepost experimental strategy that determines the quality of a DNA molecule using the relationship between the physical linkage of two assays and the genomic distance between the same two assays [ 13 – 15 , 17 ]. A milepost assay was established using plasmid pAV-CMV-GFP with CMV-Enh FAM as the anchor assay that was duplexed with different HEX assays that increased in distance from the anchor assay ( Fig 10 ).…”

“…The integrity of a DNA molecule can be evaluated with a single pair of ddPCR assays or with a milepost experimental strategy to determine the quality of a DNA template using the relationship between the physical linkage of two assays on the same DNA molecule and the genomic distance between the same two assays [ 13 – 15 , 17 ]. In this strategy, a reference assay is duplexed with assays that get progressively further away from the reference assay ( Fig 10A ).…”

“…The same non-ITR assays used for the encapsidated vector genome identity can be combined in a ddPCR-based milepost experiment to determine the vector genome integrity [12][13][14][15][16][17]. This experiment uses an anchor assay at one end of the genome that is duplexed with one of several different assays that increase in distance from the anchor assay.…”