Fig 1.
Views of glass slides (chips) for genotoyping.
A) Schematic of glass slide, hydrophobic tape, and well design. B) 24 channel chip, top view; C) 24 channel chip loaded with 72 hpf zebrafish embryos; D) Base unit; E) Base unit with loading of 24 channel chip.
Fig 2.
Flow diagram for genetic material extraction using the ZEG.
Table 1.
Results comparing chip and device designs.
Testing results comparing two different micro-abrasion chip and device designs.
Fig 3.
A) Evaluation for cross-contamination. Neighboring wells on a 24 channel chip were loaded either with an embryo or with a control (water blank); extraction was performed, followed by PCR and then HRMA. No signal was obtained in the water blank controls. Note, well B8 was designated in the software as a negative control and was therefore marked as a “Water (Negative)” with a block box; the maroon boxes labeled “Negative” were scored by the software algorithm as being negative/not amplifying. B) Representative images of cellular material collected following ZEG extraction; trypan blue staining. Scale bar 5 μm.
Fig 4.
Multi-modal analysis of genetic material collected from ZEG.
A) HRMA analysis of embryos carrying a nucleotide point mutation in the abcd1 gene. Red curves, wild-type; blue curves, heterozygous mutant; gray curves, homozygous mutant. X-axis, melt temperature (°C); y-axis, normalized change in fluorescence with temperature. B) Agarose gel electrophoresis: left, PCR for Gal4-VP16 in 74 embryos; positive results are easily scored (arrows). 2% agarose gel, four rows of lanes, 20 wells/lane, molecular weight marker at far left, final two wells in upper right are negative control and positive control. Right, PCR for abcd1zc90; scoring is indicated above each well; MW, molecular weight; +, wild-type; -, homozygous mutant; h, heterozygous; w, water control; p, positive control. C) Chromatogram sequence results from sequencing PCR products of abcd1zc90 genotyping. Example of entire amplicon sequence read shown; highlighted area shown below in higher resolution for three different genotypes (wild-type, heterozygous, and homozygous). Dotted orange line indicates start of 12-bp mutant insertion. D) 72 hpf larvae; left larva is transgenic and GFP expression is visible in the heart. E) Gal4 amplicons (red arrow) from alternating GFP+ and GFP- larvae. Gal4 amplicon is seen in every lane in which the larvae was scored positive by visible presence of GFP+ heart seen under fluorescence microscope, and no amplicon in GFP- larvae. MW, molecular weight marker.
Fig 5.
Behavior results following ZEG genotyping.
Behavior results for 7 dpf larvae, comparing embryos genotyped on the ZEG to controls; there were no statistically significant differences. Box-plot analyses.