Metagenomics: DNA Isolation from the soil sample
What is Genomics?
Why the word 'Meta'?
After finding the genome of the human being, scientists were curious to find the genomes of organisms from all domains of life. 'Meta' refers to the genome of all living organisms present in the environment. This includes archaebacteria, bacteria and eukarya, and viruses too.
What is Metagenomics?
It refers to analysing the DNA of the organisms obtained directly from the environment. To date, the approach has been applied only to microorganisms.
Why do we need Metagenomics?
Traditional microbiology is based on growing microorganisms on 'pure culture media' and then analysing their DNA. However, this method is limited to very few microorganisms, as more than 90% cannot grow in this way. Hence, we remained oblivious to the existence of a vast majority of the microorganisms present in the environment. Metagenomics determines the fate of these microorganisms under natural conditions, without growing them in the culture media. Thus, it reveals the microbial diversity and its change in the microbial ecosystem.
What environments can be analysed by Metagenomics?
Every environment from which the nucleic acids can be extracted is accessible to Metagenomics. This also includes some of the most remote and harsh environments like mines, volcanoes, radioctive, saline and acidic environments, and inside the gut, skin, mouth etcetera of an organism.
Objective:
1.
Isolate DNA from the soil sample
2. Running DNA in gel electrophoresis
Requirements:
DNeasy PowerSoil Kit
Pipettes
Tissue Roll, Discarding beaker/bucket.
Instruments requirements:
Vortex machine, Centrifuge Machine, and Incubator.
The function of the
reagents and solution:
— C1:
Sodium Dodecyl Sulphate- cell lysis, break down fats and lipids, removes humic
acid.
— C2:
Reagent- Removes non-DNA (org. and Inorg.) material, removes humic acids,
remove cell debris.
— C3:
Reagent- Removes non-DNA (org. and Inorg.) material, removes humic acids,
remove cell debris.
— C4:
High Salt concentration- Facilitate DNA binding with the Silica system.
— C5:
Ethanol-based solution- Cleanses DNA-silica binding system. Purifies DNA.
— C6:
low mM Tris- Sterile solution- Selectively releases DNA from the silica
membrane
Protocol:
1.
Add 0.25 g of a soil sample to the PowerMax Bead Tube.
[The amount of soil to process will depend on the soil type. The typical amount is 5 g, although dry soils may require less starting material (1 g) and wet soils may require more (up to 10 g). For mulch and potting mixtures, up to 2.5 g and for composts up to 5.0 g. Up to 10 g of sandy soil may be processed. ]
2.
Gently vortex to mix.
[The PowerMax Bead Tube contains a buffer that will (a)
help disperse the soil particles, (b) begin to dissolve humic acids and (c)
protect nucleic acids from degradation].
3.
Add 60µl of Solution C1 and invert
several times.
[Solution C1 contains SDS and other disruption agents
required for complete cell lysis. In addition to aiding in cell lysis, SDS is
an anionic detergent that breaks down fatty acids and lipids associated with
the cell membrane of several organisms. ]
4.
Gently vortex to mix.
[Vortexing is critical for complete homogenization and
cell lysis. Cells are lysed by a combination of chemical agents and mechanical
shaking introduced at this step. By randomly shaking the beads in the presence
of disruption agents, a collision of the beads with microbial cells will cause
the cells to break open. The
use of the vortex adapter will maximize homogenization,
which can lead to higher DNA yields.]
5.
Secure PowerBead Tubes horizontally using
a Vortex Adapter tube holder.
6.
Vortex at maximum speed for 10 min
7.
Centrifuge tubes at 10,000 x g for 30 s.
8. Transfer the supernatant to a clean 2ml collection tube.
9. The supernatant may still contain some soil particles and colours.
[The presence of carry-over soil or a dark colour in the mixture is expected for many soil types at this step. Subsequent steps in the protocol will remove both carry-over soil and colouration.]
10.
Expect 400-500 µl of supernatant. It may
still contain soil particles.
11.
Add 250 µl of Solution C2.
[Solution C2 contains a reagent that can precipitate non-DNA organic and inorganic material, including humic substances, cell debris and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.]
12.
Vortex for 5 s.
13.
Incubate at 4 0C for 5 min
14.
Centrifuge the tubes for 1 min at 10,000 x
g
15.
Avoiding the pellet, transfer up to 600 µl
of supernatant to a clean 2 ml collection tube.
[The pellet at this point contains non-DNA organic and inorganic material including humic acid, cell debris and proteins. For best DNA yields and quality, avoid transferring any of the pellets.]
16.
Add 200 µl of Solution C3 and vortex
briefly.
[Solution C3 has a second reagent to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.]
17.
Incubate at 4 0C for 5 min
18.
Centrifuge the tubes for 1 min at 10,000 x
g
19.
Avoiding the pellet, transfer up to 750 µl
of supernatant to a clean 2 ml collection tube.
20.
Add 1200 µl of Solution C4 to mix.
[Solution C4 is a high-concentration salt solution. Since DNA binds tightly to silica at high salt concentrations, this will adjust the DNA solution salt concentrations to allow binding of DNA, but not non-DNA organic and inorganic material that may still be present at low levels, to the MB Spin Columns.]
21. Vortex
for 5 s
22. Load
675 µl onto an MB Spin Column and centrifuge at 10,000 x g.
23. Discard
flow through.
[DNA is selectively bound to the silica membrane in the MB Maxi Spin Column device in the high salt solution. Contaminants pass through the filter membrane, leaving only DNA bound to the membrane.]
24. Repeat
step 21, until all of the samples have been processed.
25. Add
500 µl of Solution C5.
[Solution C5 is an ethanol-based wash solution used to further clean the DNA that is bound to the silica filter membrane in the MB Maxi Spin Column. This wash solution removes residual salt, humic acid, and other contaminants while allowing the DNA to stay bound to the silica membrane.]
26. Centrifuge
for 30 s at 10,000 x g.
[The second spin removes residual Solution C5 (ethanol wash solution). It is critical to remove all traces of wash solution because the ethanol in Solution C5 can interfere with many downstream DNA applications such as PCR, restriction digests and gel electrophoresis.]
27.
Carefully place the MB Spin Column into a
clean 2 ml collection tube.
Avoid
splashing Solution C5 onto the column.
28.
Add 100 µl of Solution C6 to the centre of
the White Filter Membrane.
[Placing Solution C6 (sterile elution buffer) in the centre of the small white membrane will make sure the entire membrane is wet. This results in a more efficient and complete release of the DNA from the silica MB Maxi Spin Column membrane. As Solution C6 passes through the silica membrane, DNA that was bound in the presence of high salt is selectively released by Solution C6 (10 mM Tris), which lacks salt.]
29.
Centrifuge at room temperature for 30 s at
10,000 x g.
Discard
the MB spin column
30.
Store the DNA at -20 0C to -40 0C
for down streaming applications.
Instructions to the teachers:
1.
The class can be divided into groups of 5-6 students.
2.
Each group should be provided with a copy of a protocol.
3.
The protocol and precautions have to be discussed by a teacher in the
class.
4.
After the discussion, the groups should be given 5 minutes to let them go through the protocol, in letting them prepare a list of
requirements, calculations, charts, tables as per the requirements, and in taking up their queries.
5. Begin the practicals.
Instructions and precautions for the students:
1. Make students read each step carefully.
2. Instruct them to follow the instructions and carry each step gently, and in an organized way.
3. Inform students to shake the solutions before adding them to the tubes.
4. Taking pictures of each step to save them for their practical records, is a welcoming step.
5. Students must be warned not to gossip, wander, linger, and use phones during the practicals.
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