How DigC Works
Using flow sorting or micromanipulation, cells are placed one-by-one on the AmpliGrid slide’s reaction sites and analyzed by DigC. Figure 1 depicts a stable (nondividing) haploid cell such as a sperm cell that contains one set of chromosomes with one chromatid each. Restriction enzymes are pipetted onto the single cell to fragment its chromosomes. Then the AmpliSpeed thermocycler heats up the AmpliGrid slide to denature the DNA. This produces two single-stranded DNA templates from each sequence, depicted as red, yellow, blue, and green fragments. A 4-plex PCR reaction mix is added and the entire volume is aliquoted into four subreactions across the AmpliGrid.
The 4-plex PCR, specific for the red, yellow, blue, and green fragments, is run and then the products are analyzed by gel or capillary electrophoresis. Since the chromatid of the haploid cell has been denatured into two strands, studying the illustrative gel bands in Figure 1 should show 0, 1, or 2 bands of each color:
• 0 if none of the fragments were amplified correctly (they were drop-outs)
• 1 band, if either there was a drop-out or both colors happened to end up in the same reaction site
• 2 bands, if both reactions worked and the fragments took part in separate reactions.
Note, then, that if all of the bands corresponding to a color across all four sub-reactions are added up and more than two bands are obtained, there has to be more than one chromosome present in the cell. The chromosome corresponding to the surplus color must have been aneuploid. The inverse, however, is not true; observing two or fewer bands does not rule out aneuploidy. A drop-out of a surplus fragment would cause a false declaration of the corresponding cell as normal. This explains why an extremely low drop-out rate is required for accurate tests based on DigC.
Figure 2 shows actual results from analyzing stable (i.e., nondividing) diploid and triploid fibroblasts from a human cell line. An 8-plex reaction was used; that is eight independent sequences located on chromosome 21 were redundantly amplified and the reaction mix across eight subreactions was divided (one gel band per reaction).
A stable diploid cell has two copies of chromosome 21 with one chromatid each, that means that after denaturing researchers expect to see a maximum sum of four copies of each genetic fragment across all eight reaction sites (Figure 2, upper gel image). If more are seen, then the cell must have been triploid. This is exactly what is observed in Figure 2 (lower gel image); the second to last band occurs five times. Thus the corresponding chromosome is diagnosed triploid.
The stochastic probability of a false negative such as not finding five or six bands for a triploid cell is 20% for this 8-plex/8 sub-PCR setup. In an analysis of 40 triploid human fibroblast cells, a false negative rate of 23% was observed, very close to the stochastic prediction.
Experimental false negative rates are expected to be slightly higher than stochastic predictions due to a small amount of drop-outs. Increasing the degree of multiplex and/or number of subreactions quickly reduces the expected false negative rate. The same 8-plex reaction spread across 12 sub-PCRs theoretically achieves <2% false negatives; increasing the multiplex degree to 24 and spreading across eight sub-reactions predicts a false negative rate <1%.
DigC has been successfully applied to Trisomy 21 detection and can be extended to diagnose other genetic disorders. By varying the multiplex grade and the number of reaction sites, test accuracy can be engineered to a wide range of specifications.