I have a little story to relate here, and I would be interested to hear back from anyone who has an idea what is happening.
It all started last summer when our lab took on a project for another lab at a different institution. The researcher shipped me their DNA in plates, plus primers and instructed me to perform multilocus genotyping on the roughly 600 samples. Upon receipt, I ran a quick PCR check of a few samples for each locus, and everything looked beautiful so I tossed the project into the freezer intending to process everything in a week or two when I knew I would have time to devote some time directly to this job. When I got back to the project, I ran the same quick PCR check just to be sure, and this time nothing really worked. Perplexed, I repeated the exercise, thinking that perhaps I forgot to add something crucial, but again the same non-result. I spent the next two weeks frantically troubleshooting this project, hesitant to contact the client since I had no idea what had happened to once perfectly good DNA that had only been opened once and had been placed in a freezer with no temperature fluctuations.
Did I contaminate the DNA with some degrading compound in the brief period I had it opened? This seemed unlikely since I do this process all the time, using the same lab practices I used during these PCR checks. Eventually I contacted the other lab and they sent me more DNA to work with. When I received that shipment, I processed all of the samples immediately in fear that they also would degrade. During this processing, I stumbled onto a bit of evidence about what may have happened. In my PCR mix, I use phenol red as a colorant. This is also a handy pH indicator which is a lovely dark red above about pH 8, but goes to an alarming yellow when the pH drops. I was doing small PCR reactions (4uL in 384well plates), so I had 3uL mastermix in a plate to which I was adding 1uL DNA. I added some DNA to a set of these reactions, and watched as they immediately changed from red to yellow. Immediately I took a few microliters of a sample and streaked it across a pH strip -- pH 5!! This prompted me to inquire to the other lab about the method used to extract the DNA and the buffer in which it was stored, etc. Samples were all extracted by the popular Qiagen kit, but this was actually done at a third lab so they weren't sure of the storage buffer. I was put in contact with the next lab, and they claimed the DNA was always eluted in Tris-Cl pH 9.0 (hey, my favorite buffer!!). I insisted this couldn't be the case and wondered if they had accidentally used nanopure water from an RO source or something that might actually have such a low pH, but they stated otherwise, and there was no use arguing anymore. I finished processing the samples and put the whole mess behind me, thinking it would always remain a nagging mystery.
In November I traveled to another lab to learn a technique for a new instrument we had received. As a part of this exercise I brought some DNA with me that I had prepared for the process, but paranoid as I can be, I decided to bring all the pieces of my chemistry along in case anything went wrong. These pieces included several plates containing PCR reactions containing phenol red. Everything traveled with me in my luggage in an insulated container with samples a bit of dry ice. I inspected the contents upon arrival, and all seemed in order, so I tossed them into the freezer. The next day, I prepared to process these samples, retrieving them from the freezer and allowing them to thaw. I was making some notes into my lab book and picked up a plate to check if it had yet thawed and was horrified to find everything had gone to yellow!
"Not again," I thought.
I frantically started looking for some Tris buffer to add to bring the pH back to where it should. Surprisingly, the lab I was in had none on hand, so I headed down the hall, bothering anyone I found in a lab for a little Tris. I located some within 10 minutes, took an aliquot into a falcon tube and headed back to my precious samples. I grabbed the first plate, and just before I tore the foil seal off, I saw the wells had gone from yellow back to red. What the hell?? Upon closer inspection, I saw this was only the case in a few of the wells that I happened to have opened briefly, and thus exposed to the atmosphere before resealing. Curious, I tore the foil seal off, put a new seal on, vortexed, and spun my plate down. Now all the wells were back to red.
So exposure to the atmosphere seemed to have solved my pH problem. So what can pH do to DNA? DNA is actually a pretty stable molecule (read up on the RNA world hypothesis for why it is so stable). It is an acid and as such, is most stable in a slightly basic buffer solution (that's why we love Tris so much). However, raise the pH too much, and the bases no longer pair (e.g. alkaline denaturation as in Illumina preps), or decrease the pH too much and other bad things start to happen. Low pH, I have read, leads to depurination (loss of A or G bases) of your DNA strands, effectively fragmenting DNA into unusable bits (no longer than about 30 nucleotides). How low does it need to be? In theory, anything acidic will contribute to this effect, but the more acidic you get, the more rapidly this will occur. If my chemistry background serves correctly, things will really start to change as you approach the pKa of DNA, which is somewhere around pH 5.0 -- right about where I had measured the pH of the DNA from the project last summer. Storing DNA in water, rather than a buffered solution is known to be less ideal than the buffer, and this may be related to the natural dissolution of carbon dioxide as carbonic acid from the atmosphere into standing water, but the pH of such water is generally measured around pH 6.7 or so, not terribly acidic at all.
So what could be happening here? When you place samples into a plate sealed with foil, there is slow evaporation/sublimation of your storage buffer over time, presumably due to slow air exchange through the adhesive layer holding you foil in place. During an average airplane flight, despite the pressurization of the cabin, everything on the plane is at a markedly lower pressure than when the plane is on the ground. The gaseous contents of the cabin aren't terribly different than what you find at sea level, otherwise there wouldn't be enough oxygen to remain conscious at 35,000 ft. So, low partial pressure of gaseous components, and subtle permeability of your sealed plate. This should actually release dissolved gases back into the atmosphere, and the loss of carbonic acid should actually raise the pH. But that's not what I saw, and everything can be fixed by thawing, removing the seal briefly, and applying a new seal upon arrival at your destination. So problem solved, but what was the problem in the first place?
Anyone??
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