Sep 13, 2023
Version 1
Extraction of high molecular weight DNA from nasal lining fluid V.1
- Samuel Montgomery1,2,3
- 1Telethon Kids Institute;
- 2University of Western Australia;
- 3Curtin University
Protocol Citation: Samuel Montgomery 2023. Extraction of high molecular weight DNA from nasal lining fluid. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qwqovo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 13, 2023
Last Modified: September 13, 2023
Protocol Integer ID: 87717
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.
Guidelines
This protocol can be used to extract DNA from both low biomass (nasal lining fluid, BALf, nasal swab) and high biomass samples (bacterial culture)
Materials
The following materials were utilised for this protocol:
- Puregene tissue kit (Qiagen, #158023) containing cell lysis solution, protein precipitation buffer, proteinase K, RNase A, and DNA hydration buffer
- Ethanol, molecular grade (Sigma, #E7023)
- MetaPolyzyme (Sigma, #MAC4L)
- Phosphate buffered saline, sterile filtered (ThermoFisher, #10010023)
- 30% polyethylene glycol (PEG) 8000 solution in 1.6 M NaCl pH 6.7 (Bioworld, #41620040-1)
- UltraPure molecular grade water (ThermoFisher, #10977015)
- GenElute linear polyacrylamide (Sigma, #56575)
- 0.1mm silica/zirconia beads (Biospec, #11079101z)
- Isopropanol, molecular grade (Sigma, #I9516)
Safety warnings
While this protocol does not utilise common hazardous chemicals used for DNA extraction (phenol/chloroform), care should be taken to follow the recommendations in the MSDS provided which each reagent, and ensure proper storage for the dangerous goods utilised in this protocol (flammable liquids etc)
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
Preparation of reagents
Preparation of reagents
MetaPolyzyme is recieved as a lypholysed powder. Reconstitute following manufacturers instructions to a final concentration of 5 mg/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. 1 mL 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
DNA extraction
1h
1h
Add 5uL of MetaPolyzyme (5 mg/mL ) to 45 µL of sample in PBS in a 1.5mL microtube
Incubate for 01:00:00 at 35 °C using a Thermomixer at 500 rpm
1h
Add sample (~50uL) to the prepared 2.0mL screw cap microtube containing beads
Add 250 µL 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 00:00:30 in the Precellys24 bead beater
30s
Place sample on ice for 00:01:00
1m
Bead beat sample for 00:00:30 in the Precellys24 bead beater
30s
Centrifuge the sample after bead beating at 10000 x g for 00:01:00
1m
Remove supernatant (~300uL) and place in a new 1.5mL microtube
Incubate for 00:30:00 at 37 °C in a thermomixer at 500 rpm
30m
Increase the temperature to 56 °C and incubate for 01:00:00 in a thermomixer at 500 rpm
1h
Briefly place samples on ice to cool down to room temperature
Add 100 µL 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 00:05:00
5m
Centrifuge tube at 16000 x g for 00:03:00
3m
Add supernatant to a new 1.5mL LoBind tube, careful not to disturb the protein pellet
DNA precipitation
DNA precipitation
2h 47m
2h 47m
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 02:00:00
2h
Centrifuge at 14000 x g for 00:30:00 at 24 °C
30m
For high biomass samples where expected DNA recovery is high
Add 1 volume of isopropanol to the sample and incubate at Room temperature for 00:05:00
5m
Centrifuge at 16000 x g for 00:05:00 at Room temperature
5m
Discard the supernatant by slowly drawing with a pipette at the air-liquid interface to avoid disturbing the pellet
Note
Pellets when precipitated with PEG will be near invisible, and easily detached from the tube, so care must be taken
Add 400 µL of 70 % (v/v) ethanol to the sample and invert several times to wash the pellet
Centrifuge at 16000 x g for 00:01:00
1m
Discard the supernatant and add 400 µL of 70%0 % (v/v) ethanol to the sample and invert several times to wash the pellet
Centrifuge at 16000 x g for 00:01:00
1m
Discard the supernatant, and allow the pellet to air dry for 00:05:00
5m
Add 30 µL of DNA hydration solution or nuclease free water to the pellet
Incubate in the fridge at 4 °C Overnight
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
Protocol references
Saladié M, Caparrós-Martín JA, Agudelo-Romero P, Wark PAB, Stick SM, O'Gara F. Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid. Front Microbiol. 2020 Oct 6;11:572504. doi: 10.3389/fmicb.2020.572504. PMID: 33123104