The demand for high molecular weight (HMW) DNA for long read sequencing has skyrocketed in the last several years. However, its extraction and handling involve careful techniques to ensure integrity of sample quality, especially when working with complex samples. Incorporating liquid nitrogen in the initial steps to stop enzymatic reactions, and during homogenization in a high throughput format, effectively minimized DNA degradation and shearing from rat liver samples. Though commonly performed on single samples, our homogenization was performed in a high throughput format. Common contaminants such as proteins and cellular matrix were removed through traditional phenol/chloroform extraction. Compared to a room temperature protocol, high throughput cryogenic grinding yielded DNA fragments larger than 23 Kb while room temperature grinding produced fragments primarily between 2 and 7 Kb. This approach simplifies the preparation of multiple samples for the isolation of DNA for long read sequencing.
The increasing number of downstream applications, especially in sequencing, relies on isolating high quality HMW DNA, which can be a challenging task. Traditional methods rely on grinding tissue cryogenically using a mortar and pestle with liquid nitrogen. A common subsequent step is to remove contaminants using phenol/chloroform extraction, a laborious and slow process, first used by Kirby (1957) to extract nucleic acids from rat liver. In addition to traditional nucleic acid extraction protocols, commercially available kits can be used following cryogenic grinding, which also follows the practice of overall gentle handling. These kits consist of a variety of mechanisms for purification, such as magnetic materials (Qiagen and PacBio), gravity-flow columns (Qiagen), and glass spheres (New England Biolabs).
Previous in-house testing was successful in isolating HMW DNA from plants using cryogenic bead beating (de Vergara, 2024). This paper builds on that research for animal tissue samples and outlines a simple combination of a liquid nitrogen-based mechanical lysis followed by a phenol/chloroform method to extract HMW DNA from animal tissue in a high throughput format. Using a 2010 GenoGrinder and AC Block cryogenic holders, up to 48 samples can be homogenized at a time. Keeping the samples frozen, which is controlled by the AC Block, resulted in powdered tissue that was suitable for subsequent enzymatic digestions and organic solvent-based cleanup. The ability to use a homogenizer translates into less manual labor and potential cross-contamination than using mortar and pestle, while allowing for higher throughput processing of samples.
Sampling: Using the CryoCoolerâ„¢, archived rat liver stored in liquid nitrogen vapor was kept frozen while 50 mg samples were cut and weighed.
Cryogenic Animal Tissue Preparation: The frozen samples of 50 mg rat liver, in triplicate, were placed in 4 ml polycarbonate vials with polypropylene lined caps, each containing one 3/8 in stainless steel grinding ball. The vials were placed in an AC Block, as part of the AC Block Kit, and chilled in liquid nitrogen for 3 minutes before bead beating. The frozen vials were then homogenized using the 1600 MiniG® at 1,500 rpm for 1 minute. The grinding balls were removed after processing. The powdered tissue was suspended in 400 µl of Homogenization Buffer consisting of 50 mM Tris, pH 9, 100 mM EDTA, 200 mM NaCl (Gibbons et al., n.d.).
Room Temperature Animal Tissue Preparation: 50 mg of samples, in triplicate, were weighed out and placed in 4 ml polycarbonate vials with polypropylene lined caps with one 3/8 in stainless steel grinding ball. Then, 400 µl of Homogenization Buffer was added. Vials were homogenized using the 1600 MiniG® at 1,500 rpm for 1 minute.
DNA Isolation Method: The homogenates were then transferred to clean 5 ml snap cap tubes. Each tube received 40 µl of 10% SDS and 40 µl of 10 mg/ml Proteinase K. The tubes were inverted to mix and then incubated at 55°C for 1 hour. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the homogenate. Samples were mixed gently by inversion, then centrifuged for 2 minutes at 12,000 rcf. The aqueous phase was transferred to a clean polypropylene snap cap tube. RNase A treatment was performed by adding 5 µl of RNase A Solution and incubating at 37°C for 15 minutes. Then, 1/10 volume of 3/5 M sodium acetate was added and mixed, and then two volumes of ice-cold 95% EtOH were carefully layered over the eluent. The tubes were then mixed and incubated at -20°C for 1 hour. The DNA solution was spun down at 10,000 rcf for 20 minutes, then the supernatant was removed. The pellet was washed twice with 500 µl of ice-cold 70% EtOH and decanted. Samples were air-dried for 20 minutes, without fully drying. The pellets were dissolved in 50 µl of TE Buffer.
Enzyme Assay: HindIII (New England Biolabs) was used to fragment the isolated HMW DNA for a comparative analysis on a BioAnalyser.
DNA Analysis: Samples were quantified using the Qubitâ„¢ Flex Fluorometer (ThermoFisher Scientific) with a QubitTM dsDNA Quantitation, Broad Range kit. DNA purity was assessed on the DS-11 FX+ Spectrophotometer/Fluorometer (DeNovix). Fragment size profiles were visualized through 1% agarose gel and microfluidic DNA chip using the Agilent DNA 12000 Kit on the BioAnalyzer (Agilent).
HMW DNA was isolated using a cryogenic method that showed fragment sizes of at least 23 kb, or larger, when compared to non-cryogenically processed samples. The heavy smearing of the non-cryogenic DNA signifies shearing when homogenizing in buffer. Both methods show significant DNA yields, as well as acceptable purities (Table 1). The agarose gels reflect trace RNA and considerable difference in fragment sizes, where the cryogenically prepared animal tissue is at least 23 kb in size, against a ladder of Lambda/HindIII digest (Fig. 1). To verify the size of the cryogenically isolated DNA, samples were digested with HindIII so they could be detected using a BioAnalyer (Agilent). Digested and undigested DNA were first checked on an agarose gel, then subsequently on the BioAnalyzer. Undigested DNA was not detected on the BioAnalyzer, with an upper limit of 17 Kb, however DNA could be detected after a HindIII digest. This validates the DNA isolated through cryogenic preparation was bigger than the upper BioAnalyzer marker of 17 Kb (Fig. 2).
Cryogenic homogenization of rat liver with the AC Block in a high throughput homogenizer yielded HMW DNA from rat liver. This supports previously generated data with maize leaves (de Vergara, 2024). Although homogenizing in buffer had a higher yield, DNA was highly sheared, in comparison to bead beating without buffer. The HMW DNA with decent yield and purity, resulting from the former process, can be used for long-read sequencing, a rapid-growing technology. An initial step of cryogenic homogenization is critical for isolating HMW DNA, with greater flexibility being allowed with subsequent purification steps, whether it be through traditional protocols, like phenol/chloroform or other kit-based protocols.
de Vergara, Faith. 2024. Isolating High Molecular Weight DNA using the Synergy Chemistry with Cryogenic Sample Preparation. From https://opsdiagnostics.com/notes/cryosyn.html
Gibbons, L., Brangs, H., & Burden, D. (n.d.). Bead Beating: A Primer. Retrieved November 4, 2024, from https://opsdiagnostics.com/notes/ranpri/OPSD_Bead_Beating_Primer_2014_v1.pdf
Kirby, K. S. (1957). A new method for the isolation of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acid and protein. Biochemical Journal, 66(3), 495-504. https://doi.org/10.1042/bj0660495