Measuring and Analyzing HMW DNA
High molecular weight genomic DNA is often viscous leading to challenges in handling, transferring, and measuring. Prior to measurement, samples should be properly homogenized following the guidelines in Homogenization of High Molecular Weight DNA (HMW DNA) Samples. Homogenization prior to quantitation is particularly important if samples have been frozen for long term storage, where gDNA is likely to be unevenly distributed upon thawing. When measuring thawed samples, allow them to reach room temperature and homogenize to enable consistent measurements.
HMW DNA samples that were prepared using high agitation speeds during lysis are suitable for concentration and purity assessment on microvolume spectrophotometers (MVS) (e.g., Nanodrop®) after homogenization. These samples should be briefly vortexed to ensure even distribution of the gDNA in the solution before quantitation; a short vortex will not shear DNA. As the Monarch HMW DNA Extraction Kit protocol efficiently removes RNA, 260 nm absorbance values provide an accurate indication of the amount of DNA present. Fluorometric measurement of HMW DNA tends to underestimate DNA concentration and should be used with additional considerations described below.
Pipetting and concentration measurement of DNA isolated using low agitation speeds (UHMW DNA) is challenging and often highly imprecise due to extreme sample viscosity. A commonly recommended method for UHMW DNA concentration measurement is taking 1 or 2 µl aliquots from top, middle, and bottom of the solution and using the average concentration value. However, this approach produces highly variable results. Fluorometric measurement of UHMW DNA is specifically inaccurate given that the extreme viscosity causes some sample to be pulled out of the pipette tip upon removal.
Since HMW DNA <250 kb is easy to handle and can be accurately measured using standard spectrophotometric methods, UHMW DNA measurement can be made more accurate by reducing the overall DNA fragment size of a small aliquot of the sample. In “A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA”, a methods paper published in Biotechniques, we outline a simple and effective approach for shearing small aliquots of UHMW DNA samples to a size conducive to accurate spectrophotometric measurement. Essentially, the approach is as follows:
- Using a P200 pipette tip, preferably low retention, pull 5-10 µl of a homogenized UHMW DNA sample into the pipette tip.
- Expel the sample from the tip, and then pull up the sample again to ensure the aspired volume is accurate.
- Scrape the tip across the bottom of the tube to break the threads of DNA
- Transfer to a round bottom 2 ml microfuge tube
- Add one borosilicate glass bead, either 4 mm (NEB #T3005) or 3 mm (Sigma # Z143928).
- Vortex at maximum speed for 1 minute in pulses of 5-10 seconds. This will effectively reduce DNA size to <150 kb, enabling accurate measurement.
- Pulse-spin in a benchtop mini centrifuge to collect sample
- Transfer the sample to a new 1.5 ml microfuge tube, preferably DNA low bind. If starting with 10 µl, expect to recover about 8-9 µl, as some volume will be retained on the bead.
- Vortex briefly to mix, and measure on spectrophotometer.
When measuring HMW DNA, Qubit-based concentration values are consistently >25% lower than the OD-based. When using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), we have found this underestimation to be a result of the standard used in the assay. The supplied standard DNA for the calibration curve in this kit is Lambda DNA, and in our experience, this standard may not be suitable for the quantitation of HMW DNA. Exchanging the 100 ng/µl standard with commercial genomic DNA derived from Jurkat cells (Thermo Fisher Scientific # SD1111) reduces the difference with the OD-derived values to 6.5 %. LC-MS analysis revealed that this gDNA standard contains 4.8% RNA, providing an explanation for another 5% difference between both methods. Taken together, the shearing protocol described above is compatible with fast and inexpensive absorbance measurement (e.g., Nanodrop). If purity ratios indicate that no significant RNA or other contaminant is present, that method can be used. But where fluorescence (“Qubit”) measurements need to be used, for example for quantitation of nanopore libraries, the use of a high-quality gDNA standard, preferably RNA-free, will enable accurate measurements.
Storage of HMW DNAThe Monarch gDNA Elution Buffer II (NEB #T3056) provided with the kit (10 mM Tris-HCl, pH 9.0, 0.5 mM EDTA) was developed as a long-term storage buffer. The combination of EDTA and high pH provides optimal protection against nucleases. If the sample will be actively used, it is recommended to store HMW samples at 4°C; for long term storage, store at -20°C. Avoid repeated freeze thawing and always use low bind tubes to prevent DNA from binding to the tube walls.
- Monarch HMW DNA Extraction Kit for Cells & Blood
- Monarch HMW DNA Extraction Kit for Tissue
- Monarch HMW gDNA Elution Buffer II
- Monarch HMW DNA Extraction Kit for Cells & Blood Product Manual
- Monarch HMW DNA Extraction Kit for Tissue Product Manual
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits
- ’ Ultra-Long DNA Sequencing Workflow®Guidance for HMW DNA Extraction Upstream of Oxford Nanopore Technologies
- Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow