Fig 1.
Schematic of formalin-induced crosslink formation and the removal of crosslinked proteins by treatment with proteinase K.
(A) A protein amine can nucleophilically attack the formaldehyde carbonyl to yield an iminium ion, which can then react with another primary amine from DNA, RNA, or proteins to form a crosslink. This crosslink reaction is in reversible dynamic equilibrium [10, 21]. (B) Treatment with a protease, proteinase K, allows free DNA (fDNA) recovery. Here, Nuc designates an amine nucleophile from the DNA.
Table 1.
Current methods for formaldehyde crosslink removal & DNA recovery from formalin-fixed specimens.
Fig 2.
Schematic of the VFD-mediated fDNA recovery technique.
(A) The protocol begins with Vortex Fluidic Device (VFD) treatment (7 krpm, room temperature, abbreviated RT, 1 h) of a mixture of proteinase K and the frozen, then broken-up tissue. The reaction mixture is next processed to remove solids and DNA polymerase inhibitors. The recovered fDNA is then purified and concentrated. Finally, the DNA is amplified, quantified, and characterized by (B) qPCR and (C) DNA sequencing of the samples. Larger versions of panels B and C are provided in S1 and S2 Figs. Threshold cycle (Ct) and endpoint fluorescence values are given in S1 Table.
Table 2.
PCR primer sequences and annealing temperatures.
Fig 3.
Quantification for optimizing the VFD rotational speed for fDNA yields.
After proteinase K and VFD treatment at the indicated speeds, (A) absorbance at 260 nm and (B) the ratio of absorbances 260:280 nm quantifies DNA and protein yields, respectively, with positive and negative controls. Full UV-vis spectra for these samples are shown in S3 Fig. SYBR Green I fluorescence-quantified (C) dsDNA concentration and (D) fold increase in dsDNA yield between non-VFD-processed and VFD-processed samples. The negative control indicates samples not subjected to VFD processing. Buffer only controls lacked lobster tissue. The positive controls included DNA that had not been formalin fixed. Additional controls demonstrated a conventional method and an intermediate method (processing time of the conventional method and temperature of the VFD-mediated method) used to process fixed and fresh tissue without the VFD. The error bars designate the standard deviation for sample measurements at the indicated condition (technical replicates, n = 3).
Fig 4.
Amplification of an 183-bp fDNA target from the ATP synthase gene of the lobster mitochondrial genome.
(A) Quantitative PCR and (B) agarose DNA gel electrophoresis identified 7 krpm as the optimal VFD rotational speed for qPCR amplification. Threshold cycle and endpoint fluorescence values are provided in S2 Table. The variable-rotational speed PCR reactions were compared to a no template control (NTC), a fresh lobster DNA positive control (+), and a non-VFD-processed negative control (–). (C) The 7 krpm VFD-processed qPCR product (*) was subjected to Sanger sequencing; a mutation (G2728A, GenBank No. HQ402925) was observed (highlighted).