Hello everyone
I encountered this issue while extracting DNA from mollusc tissues with a salt-precipitation protocol. For some samples, the pigments contained in the tissues (which were originally black, grey, red) remained in the fraction containing the nucleic acids during the whole procedure. The pellets of DNA resulted coloured at every step, and the final elution maintained the same color. In my case the final elutions were of different shades of yellow, coffee-like brown, and red (always very bright), but I heard of similar cases with other organisms where the final elution was green.
I don't know the nature of these pigments in my animals (i.e., what kind of molecules they are), and all I can think about is that they may at least have the same polarity as DNA since they move in the same way during the extraction procedure.
Do you know of some possible negative influence of these pigments in downstream analyses? If yes, is there a way to separate them from the extracted DNA? For now, I did not see clear effects on quantification with a spectrophotometer and in PCR (colored samples behaved similarly to clean ones), but I don't know if in qPCR the pigments may alter the fluorescence signal.
Have you ever encountered similar issues? If yes, I will welcome every comment you can give me! Thank you!
I too have had coloured pigments co-isolate with my DNA from mollusc tissue. And likewise, regular PCRs worked well and spec results did not seem abnormal.
For qPCR might you try doing an absorption spectra somehow to see if your coloured pigment produces a peak within a wavelength that might influence your qPCR results?
Hello Davide,
Yes this is a common enough problem that several companies have created kits to deal with co-extracting molecules, often pigments. The simple answer, as you have already alluded to, is that it depends on the downstream enzymatic reaction you want to perform. Standard PCR is quite robust these days, and in general you can get amplification even with a few inhibitors in the mix (dilute the template added for instance). However there are many more sensitive downstream reactions you may want to perform. Enzymatic digestions, ligations, and yes qPCR will be more sensitive to contaminants than a PCR. Specifically, you will see that some of these contaminants will drastically skew the concentration inferred using a spectrometer. They often absorb wavelengths in the same range as nucleic acids. The best way to test whether you are experiencing this is to take a reading using an Implen or Nanodrop and then take a second reading using a fluorimetric approach (e.g. Qubit). The fluorescent dye used in Qubit quantification will only bind and fluoresce to double stranded DNA meaning the quantification you get will be more robust to contaminating compounds. The rule of thumb that I use is that if the Nanodrop reading is less than 20% higher than the Qubit reading (e.g. Nano drop/Implen = 50ng/ul and Qubit = 40ng/ul), your DNA is pretty good for most applications. However for highly sensitive downstream applications like PacBio sequencing (or qPCR depending on the nature of the contaminant) you will want these numbers to match more closely. Since I work on Fungi, this is a common problem for us (many different pigments in the cell wall) so I use a MoBio PowerClean DNA cleanup kit to get rid of these co-purifying contaminants. These kits are pretty easy to use and fast but they are expensive and you often lose a lot of DNA in the process. However for PacBio, they are the best solution we have found. Typically after cleaning up a sample with these kits I get almost the exact same reading with Qubit and Implen with pristine 260/280 ratios. I was dubious at first but it has consistently worked for us. Unfortunately the chemistry involved in these kits is proprietary, so we don't have a "home made" solution yet. Hope this helps.
dv
It is a serious problem in Cnidaria, largely because it inhibits the PCR. The solution we found is to use magnetic beads extraction kit, it greatly reduces the amount of pigment in the extraction result.
Cheers
Sergio
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