Extraction of high molecular weight DNA from nasal lining fluid
Samuel Montgomery
Abstract
Assessing the microbiome in respiratory samples is often difficult due to the low biomass of microbes often present in these samples. While there are published methods for efficient extraction of DNA from samples such as bronchoalveolar lavage fluid for 16s rRNA sequencing or metagenomic sequencing (Saladie et al., 2020), these methodologies are usually optimised for traditional short-read based sequencing technologies. With the advent of accessible and affordable long-read sequencing technologies for full length 16s rRNA sequencing (PacBio) and whole metagenomic sequencing (Oxford Nanopore), there is increased importance in extracting high quality, high molecular weight fragments of DNA from metagenomic samples. This protocol can be used to extract DNA from both low biomass (nasal lining fluid, bronchoalveolar lavage, nasal swabs) and high biomass (pure bacterial culture) samples.
Before start
This protocol extracts DNA from samples stored in 45μL of PBS - if the sample is in a larger volume, the volumes of all following reagents can be scaled up, ensuring consistent ratios throughout the protocol. Otherwise, centrifuge sample at high speed for ~5 minutes and remove supernatant, resuspending pellet in 45uL of PBS
Steps
Preparation of reagents
MetaPolyzyme is recieved as a lypholysed powder. Reconstitute following manufacturers instructions to a final concentration of 5mg/mL, aliquot into 0.6mL microtubes, and store at -20ºC until use
Create a 70% (v/v) solution of molecular grade ethanol with UltraPure water (approx. 1mL per sample)
Add a small volume (up to the line of the conical portion on the tube) of 0.1mm silica/zirconia beads to a 2.0mL screw cap microtube suitable for bead beating (one per sample)
DNA extraction
Add 5uL of MetaPolyzyme (5mg/mL) to 45µL of sample in PBS in a 1.5mL microtube
Incubate for 1h 0m 0s at 35°C using a Thermomixer at 500rpm
Add sample (~50uL) to the prepared 2.0mL screw cap microtube containing beads
Add 250µL of Cell Lysis Solution containing 0.6% (v/v) Proteinase K and 0.6% (v/v) RNase A solution to the sample
Bead beat sample for 0h 0m 30s in the Precellys24 bead beater
Place sample on ice for 0h 1m 0s
Bead beat sample for 0h 0m 30s in the Precellys24 bead beater
Centrifuge the sample after bead beating at 10000x g,0h 0m 0s for 0h 1m 0s
Remove supernatant (~300uL) and place in a new 1.5mL microtube
Incubate for 0h 30m 0s at 37°C in a thermomixer at 500rpm
Increase the temperature to 56°C and incubate for 1h 0m 0s in a thermomixer at 500rpm
Briefly place samples on ice to cool down to room temperature
Add 100µL of protein precipitation solution containing 1% (v/v) GenElute linear polyacrylamide to the sample and mix thoroughly by inverting the tube 25 times
Incubate samples on ice for 0h 5m 0s
Centrifuge tube at 16000x g,0h 0m 0s for 0h 3m 0s
Add supernatant to a new 1.5mL LoBind tube, careful not to disturb the protein pellet
DNA precipitation
For low biomass samples where expected DNA recovery is low
Add 1.5 volumes of PEG solution to the sample and incubate in the dark at Room temperature for 2h 0m 0s
Centrifuge at 14000x g,0h 0m 0s for 0h 30m 0s at 24°C
For high biomass samples where expected DNA recovery is high
Add 1 volume of isopropanol to the sample and incubate at Room temperature for 0h 5m 0s
Centrifuge at 16000x g,0h 0m 0s for 0h 5m 0s at Room temperature
Discard the supernatant by slowly drawing with a pipette at the air-liquid interface to avoid disturbing the pellet
Add 400µL of 70% (v/v) ethanol to the sample and invert several times to wash the pellet
Centrifuge at 16000x g,0h 0m 0s for 0h 1m 0s
Discard the supernatant and add 400µL of 70%% (v/v) ethanol to the sample and invert several times to wash the pellet
Centrifuge at 16000x g,0h 0m 0s for 0h 1m 0s
Discard the supernatant, and allow the pellet to air dry for 0h 5m 0s
Add 30µL of DNA hydration solution or nuclease free water to the pellet
Incubate in the fridge at 4°C
After this step, DNA is ready to be quantified and used downstream. DNA resuspended in water should be stored at -20°C, while DNA resuspended in DNA hydration solution can be stored for up to one month at 4°C
