I thought this would be a good week to help set the record straight on some popular misbeliefs about quantifying DNA (and RNA) yields and purity and especially when working with environmental samples like soil.

Problems achieving high yield and purity are exaggerated in environmental samples because of the added complexity of  microorganism lysis and inhibitor removal.  Quantifying the nucleic acids in these samples is the easy part. But if you don’t know what to look for, you can easily make mistakes in interpreting the results, which can lead to a lot of repeat work or missed critical information in your experiments.

Let’s discuss some of the common misconceptions surrounding DNA isolation and quanification and what problems to look out for before going to the next step with your sample.

1. True or False:  A higher UV A₂₆₀ reading means more DNA.

False.  A high A₂₆₀ reading does not always mean high genomic DNA yields.  One of the main reasons for a high A₂₆₀ reading that does not correlate to genomic DNA is the absorbance of UV due to highly degraded DNA or RNA. Degraded RNA absorbs a high level of UV and results in a boost to the A₂₆₀ reading. The method used to purify the DNA after lysis will determine what is present in the final sample. Many purification methods do not separate the small DNA and RNA from the high molecular weight gDNA. The PowerSoil DNA Isolation Kit does.

The only way to know what you really have in your sample is to run 5-10 µl on an agarose gel. This will give you a clear indication as to whether you have predominantly genomic DNA or a mix of nucleic acids sheared to varying lengths.

2. True or False:   Bead beating always gets higher yields of DNA

False. Not all samples need to be homogenized with high velocity bead beaters. Typically for RNA extraction of tissues and plants, definitely use the strongest method available to you. You want to break the genomic DNA down in size. For genomic DNA from a variety of environmental samples, gentle methods such as vortexing with beads will isolate high molecular weight DNA. When measuring the yields on a spec, as mentioned above, the more sheared the DNA, the higher the absorbance reading. This does not mean more DNA was isolated.  When qPCR is performed, less DNA will be added to the reaction due to the false high reading, leading to higher Cq values and inaccurate quantification in the sample.

For microbial DNA from soil, the bead beater will tend to do more damage than good. For DNA from fungus and spores, a thorough discussion on ways to optimize the lysis using different beads and heat has been described in detail. We also give some recommendations for using the bead beater and soil based on published references.

The best approach to ensure the integrity of the DNA is to run a gel in addition to the Nanodrop or UV scan so you can make a better assessment of what you really have. If you see a smear on your gel, then the bead beating was too hard.

3. True or False:  If the sample looks clear, it is free of humic and fulvic acids

False. Humic acids give the sample the characteristic brown color so if your DNA elutes with color, you know you’ve got a lot of contamination. Even if it looks clear, there can still be low levels of humic and fulvic acid or even polysaccharide contamination in the sample. Using a kit with Inhibitor Removal Technology such as PowerSoil, PowerWater, and coming soon, PowerBiofilm, will ensure that a clear eluate is actually clean.

In addition to the A₂₆₀ reading for yields, a low 260/230 ratio can be indicative that the sample still has some organic contaminants. A ratio above 1.5 is ideal. A ratio below 1.0 has significant contaminants present that may interfere with enzymatic applications.

4. True or False: If my soil has low amounts of DNA, I need to try stronger methods for lysis

False. If your soil has low amounts of DNA, you need to start with more.

The yield in soil varies greatly but for a rich organic soil with a high microbial load, yields of DNA will range between 20-30 µg per gram of soil (5-7 µg/ PowerSoil prep). According to Whitman et. al (1998),  rich top soil contains 1-2×10⁹cells/gram of soil (1).  Using an E.coli genome as an example, this equates to 5-10 µg of DNA in a gram of soil or around 1-3 µg of DNA per PowerSoil DNA Kit prep. Eluted in 50 ul, the concentration to expect for microbial rich soil is around 20-60 ng/µl. If the genome of the organisms in soil are double the size of E.coli, then yields of 40-120 ng/µl are in the correct range for microbe dense soil.

Most of the soil we use in our lab are not at the high end of microbial load, so yields of 10-20 ng/ul are not uncommon for average soil preps.

Given this information, if another isolation method gives you yields far above this range, it is not all DNA. The DNA is either contaminated with UV absorbing PCR inhibitors or mostly degraded RNA. Either way, the information obtained from genotyping will not be as complete or accurate as clean pure microbial DNA obtained from using PowerSoil and UltraClean Soil Kits.

Make sure to always check the yields on a gel and for even greater accuracy, use qPCR.

 5. True or False:  PCR is the best way to check for inhibition

True. The only way to know if the sample is inhibitor free is to use it in an enzymatic reaction. Even better is to use qPCR and perform serial 10 fold dilutions and check the efficiency of amplification using a primer pair for 16s rDNA. It is likely that there will be some background amplification in the water control because most PCR mixes have background bacterial DNA, but the difference in Cq value between the samples and the control will be far away enough to not matter (usually 6-10 cycles).

With qPCR, the desired result is a change of ~3.3 cycles between each 10-fold dilution. This indicates perfect doubling each cycle and is a sign that the sample is inhibitor free. What you may see is the first sample (undiluted) is shifted to the right and then the rest of the samples fall into place. This indicates that there is some inhibiting substances in the DNA. Remember not to add too much DNA. 1-2 µl per 50 ul PCR is adequate or around 10-100ng for the first sample and then dilute from there.  If the first sample amplifies to soon (and falls in the baseline for the instrument, where fluorescence is subtracted out as background), it will cause some problems with the standard curve so you may want to start with 10 ng and dilute from there.

Another good approach is to set up a PCR reaction that always works (such as for a plasmid) and then spike in 1 µl of the DNA from the environmental sample. If it causes the PCR to fail, or reduces the amount of product, it indicates inhibition.

Summary

These technical tips do apply to more than just soil and water samples. Even DNA from blood or tissues can be affected by inhibitors causing problems with absorbance readings and inaccuracies in quantification. The best approach is to always run a quick agarose gel to go with your Nanodrop results so you can see the integrity and composition of the sample along with the yield. Additionally, PCR or qPCR can help get a more exact quantification of the amount of gDNA or number of microbes in the starting sample.

Thanks for reading, and as always, send us your questions and comments either here on the blog or at technical@mobio.com.

Have a great week!

Suzanne

References:

1. Prokaryotes: the unseen majority.
Proc Natl Acad Sci U S A.
1998 Jun 9;95(12):6578-83.

This entry was posted on Sunday, January 31st, 2010 at 11:48 pm and is filed under Tech Tips. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.