The overall goal of this procedure is to retrieve DNA from active microorganisms that have consumed a growth substrate of interest without the prerequisite of laboratory cultivation. This is accomplished by first incubating an environmental sample with a stable isotope labeled substrate of interest under conditions that are similar to those found in the native environment. The second step of the procedure is to extract total DNA from this isotope labeled sample.
The third step of the procedure is to subject this DNA to density gradient ultracentrifugation in cesium chloride. The final step of the procedure is to fractionate the density gradient to obtain heavy and light DNA for subsequent molecular characterization. Ultimately, results can be obtained that reveal the identity of active yet uncultured microorganisms involved in the metabolism of a particular substrate through a variety of possible molecular techniques, including fingerprinting, microarray, hybridization, clone, library analysis, and metagenomics.
Hello, I'm Josh Neufeld from the Department of Biology at the University of Waterloo. I'm Eric Dunford, a graduate student in the Newfeld Lab. Today we'll be showing you a procedure for DNAS isotope probing, or DNA sip, which has become a widely used technique for linking the identity of unknown and potentially uncultivated microorganisms with their ability to use a growth substrate of interest.
We use this procedure in the Newfeld lab to study the metabolism of carbon sources in a variety of terrestrial and aquatic environments. Today we'll be showing you the application of a particular carbon substrate 13 C glucose to a soil sample. But we acknowledge that since this method was first developed by Colin Merle's Group at the University of Warwick, researchers have diversified the protocol to be applied to a variety of different environments and also using a variety of different labeled isotopes like nitrogen and oxygen.
Today we'll be focusing on carbon, but the protocol itself can be applied to different experimental designs. So let's get started. Prior to the start of this procedure, prepare all DNA stable isotope probing or DNA CYP reagents as described in the written portion of this protocol.
DNAC begins with sample incubation. Incubation conditions vary widely depending on a variety of sample and substrate characteristics. Once appropriate incubation conditions have been determined empirically, add the appropriate substrate amendment to the environmental sample.
You may also choose to include supplemental nutrients to enable microbial growth if these are required based on test incubations. Once the sample incubation has been set up, cap and seal the sample, then incubate the environmental samples containing labeled substrate. The example being shown involves the addition of a carbon 13 labeled glucose solution to a soil sample.
Next, set up a control incubation to serve as a comparison to confirm apparent labeling of nucleic acid observed in subsequent steps. Prepare this sample under identical conditions except using unlabeled carbon as a substrate cap, seal and incubate the environmental sample with unlabeled substrate under the same conditions following the incubation extract, DNA from the microcosms using an extraction protocol that is suitable for the desired downstream applications. Then quantify the extracted DNA for the following ultracentrifugation steps using aous gel and a spectrophotometer.
Use vertical or near vertical while rotors for ultracentrifugation to ensure maximum possible separation of light and heavy DNA. The Beckman Coulter VTI 65.2 rotor has 16 wells for holding 5.1 milliliter quick seal polymer tubes to prepare samples for ultracentrifugation. First, calculate the volume of extracted DNA that is required to add 0.5 to five micrograms of DNA to the ultracentrifuge tubes.
Using the previously determined DNA concentrations. Then determine the exact density of the cesium chloride stock solution using a digital refractometer. As suggested in the written protocol, calculate the volume of gradient buffer DNA solution required to generate an appropriate mixing ratio.
Using these calculations, combine the extracted DNA with gradient buffer and 4.8 milliliters of cesium chloride in a sterile disposable 15 milliliter tube. The resulting volume will be greater than the specified capacity of the centrifuge tubes in order to completely fill the tubes. Finally makes the solution by inverting the tube 10 times variations of this protocol.
Use a flora force such as ahy and bromide within the gradient solution to enable DNA to be visualized within the tube. Altering the density of DNA when prepared with pure culture DNA, including an AUM bromide gradient as shown here, is particularly useful to provide immediate visual confirmation of gradient formation following each centrifuge. Run steps in using such an approach are described in the written protocol.
To set up the ultracentrifugation first, use a bulb and passe a pipette to carefully fill the ultracentrifuge tubes with the gradient solutions. Label the tubes on the tube shoulder with a fine permanent marker. Ensure that the tubes are filled exactly to the base of the tube neck as insufficiently filled tubes have a tendency to burst during ultracentrifugation.
Once all the required tubes are filled with the sample solutions, record the precise mass of each tube sort tubes as closely matching pairs and add or remove my new quantities of solution until they're balanced to within 10 milligrams. Keep the solution level as close to the base of the tube next as possible. Then seal the tubes using a tube topper according to the manufacturer's instructions.
Check that the tubes are sealed properly by inverting them and applying moderate pressure. Weigh the tubes again to check that they're still balanced after sealing prior to ultracentrifugation. Carefully inspect each rotor well to ensure they're clean and free of any dust and debris that could puncture the tubes During ultracentrifugation.
Insert the tubes into the rotor, placing the balanced pairs opposite each other. Record the rotor location of each sample as the ultracentrifugation process can render marker labels legible. Carefully seal the rotor wells as indicated by the manufacturer.
Once the rotor wells have been sealed, load the rotor into the ultracentrifuge. Close the ultracentrifuge door and apply a vacuum for the VTI 65.2 rotor. Set the rotation speed to 44, 100 RPM the temperature to 20 degrees Celsius and the ultracentrifugation time to 36 to 40 hours.
Ensure the vacuum is on select maximum acceleration and turn off the brake to ensure the gradient is not disrupted by deceleration. Immediately following ultracentrifugation, remove the rotor carefully. Be careful not to tilt or bump the rotor to avoid disturbing the gradients within the tubes.
Finally, gently remove the tubes for gradient fractionation. DNA can be extracted from the ultracentrifuge tubes using the fractionation technique. To begin fill a sterile 60 milliliter syringe with sterile distilled and deionized water containing sufficient propofol blue dye.
To provide a dark blue color, place the syringe on the loading arm of the syringe pump. Then attach pump tubing fitted with a 23 gauge one inch needle and turn on the pump until some water comes through the end of the needle. Avoid any air bubbles in the water supply as they will negatively affect the fractionation process.
For each sample, prepare 12 sterile 1.5 milliliter micro centrifuge tubes with labels indicating the sample number and fraction. Next, fix one of the ultracentrifugation tubes to a clamp stand with a quick and controlled motion. Pierce the bottom of the tube along the tube seam using a fresh 23 gauge one inch needle and rotate the needle to ensure a uniform hole has been formed.
You may find that latex gloves provide better grip on the needle shaft than nitrile gloves. Using the needle that is attached to the pump tubing pierce the top of the tube on the upper tube shoulder along the seam in a quick and controlled manner. Note that piercing the top of the tube followed by the bottom of the tube can also be done.
Also, note that a small amount of mineral oil coating the shoulder of the tube can help prevent leaking from an improper seal around the upper needle with a previously calibrated pump rate. Pump the dyed water into the top of the tube to yield 12 425 microliter fractions in 12 minutes. Use a timer to collect the gradient solution in the labeled micro centrifuge tubes.
Finally, check the density of all fractions for at least one sample or control gradient. Using a digital refractometer. Expect values in the range of 1.690 to 1.760 grams per milliliter with a median density of approximately 1.725 grams per milliliter to precipitate DNA from all fractions.
First, add 20 micrograms of linear poly acrylamide as a carrier for precipitation and mix by inversion. Then add two volumes of PEG solution and again, mix by inversion. Leave the tubes at room temperature for two hours to allow the DNA to precipitate following incubation.
Placed the samples in a micro centrifuge with the tubes oriented in the same direction to ensure consistent localization of the pellet centrifuge. The tubes at 13, 000 G for 30 minutes after centrifugation retrieved the samples from the micro centrifuge, then carefully aspirate and discard the supernat app. Pellet should be visible at this stage with the aid of a bright light source.
Wash the pellet with 500 microliters of 70%ethanol after washing the pellet centrifuge. The samples at 13, 000 G for 10 minutes following centrifugation carefully aspirate and discard the supernat. The pellet will be more visible now, but will dissociate from the tube wall more easily.
Allow the pellet to dry at room temperature for 15 minutes. Once pellets have dried, suspend each pellet in 50 microliters of TE buffer. Finally, run five microliters of each fraction on an aeros gel to characterize the gradient fractions using methods discussed in the written protocol.
Typical DNA SIP results will demonstrate a separation of labeled and unlabeled DNA in the gradient form by ultracentrifugation. Ideally, there'll be a complete resolution of high molecular weight genetic material such as carbon 13 and nitrogen 15 from unlabeled material in particular, unique PCR based fingerprints associated with fractions four through eight of stable isotope incubated samples, but not with native substrate. Incubated controls provide strong evidence linking specific organisms with the metabolism of particular labeled substrate when performed on DNA from two pure cultures, carbon 13 labeled methyl occus capsulitis strain bath and unlabeled Sian medi A TCC 10 21, labeled an unlabeled genomic DNA, separate into respected gradient fractions with differing densities.
In this example, heavy isotope labeled DNA can be observed in fractions four through five, whereas unlabeled DNA is found at high concentrations in fractions nine through 10. PCR amplified DNA from the same fractions were run using denature and gradient gel electrophoresis or DGGE and generated discrete banding patterns corresponding to the two organisms included in the gradient. The density of the fractions range from approximately 1.580 to 1.759 grams per milliliter, and they're shown in order of decreasing density from left to right.
Conversely, the separation of isotopes and environmental sample incubations may be more difficult to interpret. Tundra soils from Resolute Bay Canada were incubated with either carbon 12 or carbon 13 labeled glucose for a 14 day period at 15 degrees Celsius. The aeros gels of the purified gradient fraction DNA revealed smear genomic DNA across fraction seven through 10 for both carbon 12 and carbon 13 incubations.
However, when DGGE of 16 s ribosomal RNA genes was used to determine the carbon 13 enrichment of biomass from particular microbial taxa, the carbon 12 glucose incubated soil, DNA generated similar patterns across all gradient fractions while the carbon 13 glucose incubated sample generated DGG fingerprints that are uniquely associated with fractions five through eight, the conserved bands indicated by the arrows represented dominant phylo type consistent across all gradient fractions. The phylo type shifts to heavier fractions for DNA obtained from carbon 13 glucose incubated soil. The identity of this particular 16 s ribosomal RNA gene can be determined to guide subsequent metagenomic or cultivation based approaches.
We've just shown you how to perform A DNA stable isotope probing experiment from incubating samples to obtaining labeled DNA for molecular analysis. When performing this procedure, it's important to consider your experimental design carefully be careful to avoid adding too much substrate in order to avoid biasing your experiment towards fast-growing organisms. You also want to avoid extended incubation so that substrate doesn't dilute to other members of a community through a food web.
So you want to use just the right amount of substrate and just sufficient incubation times to allow you to detect small amount of labeled DNA in heavy fractions with sensitive PCR based applications. So that's it. Thanks for watching and good luck with your experiments.
