DNA Amplification Using Body Heat, No Instrument Required

Posted on by Sara Klink

Cartoon by Ed Himelblau
Cartoon by Ed Himelblau Copyright Ed Himelblau.
When I was in the lab, there was more than a few times I held tubes in my hand (maybe even under my arm) to make them thaw faster, especially reaction buffers. However, I never considered whether this could be a strategy for actual incubation although humans run at about 37°C and many restriction enzyme reactions proceed most efficiently at 37°C. But research published in PLOS ONE by Crannell, Rohrman and Richards-Kortum took this idea and decided to experiment with the possibility of eliminating an instrument-based DNA amplification.

Why not consider the quirky idea of using the human body as an incubator? Reagents are inexpensive compared to instruments, and many places in the world are resource limited. Therefore, a workable alternative to an expensive incubator that needs electricity would be useful. While PCR is the standard for amplification of DNA, a single-temperature recombinase polymerase amplification (RPA) offers many advantages compared to PCR in countries where resources are limited. This method amplifies DNA in as little as 5 minutes, reagents can be shipped lypophilized without the need for cold storage, the reaction tolerates sample impurities and functions over a range of temperatures including 37°C. In this study, the authors tested how various locations on the human body would work for incubating RPA reactions to detect HIV-1 DNA. Detection was performed using lateral flow strips, a method commonly used for store-purchased pregnancy tests.

First, the temperature of four possible body locations were measured using a thermocouple probe inside a 2ml microcentrifuge tube filled with 50µl of water (mock reaction). These body locations included inside a closed fist, a rear trouser pocket, under the arm (axilla) outside of clothing or taped to the abdomen underneath clothing. The mock reaction tubes were incubated on five different volunteers and temperature measured for 45 minutes. Within three minutes the tubes at all locations reached 31°C, the temperature needed to amplify DNA to detectable levels, but the underarm was chosen as the location for additional testing because the average temperature was closest to 37°C, the temperature recommended for RPA. Then Crannell, Rohrman and Richards-Kortum considered how to keep the tube under the arm for incubation: A 10cm wide bandage, a 5cm wide elastic sweat band or an 8cm wide strip of cotton cloth (African chitenje fabric). Again, five different people incubated mock reaction tubes using each of the three methods to keep the tube under the arm. There was little difference in temperature or time to reach maximum temperature so the researchers chose cotton cloth to continue the testing because it was inexpensive and easy to find in places that might use this method.

How does ambient temperature affect the temperature of tubes under the arm? Researchers had five volunteers incubate mock reaction tubes under four different conditions: A 4°C cold room, a 10°C cold room, room temperature and in the Houston, TX, summer sun. The ambient temperature did affect the temperature of each of the reactions. For example, the 4°C cold room yielded an average reaction temperature of 25°C while the sunny summer day in Texas averaged a 39°C reaction temperature. The room temperature and the summer day still had feasible RPA temperatures but 4°C ambient temperature was not ideal for DNA amplification.

Now that the body location, ambient temperature and method of securing tubes had been established, how did these parameters perform with RPA of HIV-1 DNA? Ten volunteers incubated reaction tubes with 0, 10 or 100 copies of HIV-1 DNA placed inside plastic zipper bags either in an office or lab (room temperature) or in a 10°C cold room. Control RPA reactions were incubated in a heat block at 37°C, 10 reactions for each ambient temperature experiment. For all reaction conditions, the 0 copies were negative for HIV-1 DNA and the 10 and 100 copies were positive by lateral flow strip with two exceptions. One room temperature incubation of 100 copies from one of the ten human volunteers was negative; the other negative result cam from one of the ten control reactions incubated in the heat block in the 10°C cold room was negative. There was no detectable difference in signal-to-background ratios for human-incubated reactions at 10°C or room temperature.

Can the human underarm be used as an incubator for recombinase polymerase amplification of HIV-1 DNA? These experiments demonstrate yes, it can. While not the most comfortable position to be in (please place these tubes under your arm and hold for 20–30 minutes), this offers people with limited access to instrument and electricity an option for using RPA in the field. And now you can say, the underarm offers more than a place to incubate your unique bacterial microbiome.

Reference
Crannell, Z.A., Rohrman, B. and Richards-Kortum, R. (2014) Equipment-free incubation of recombinase polymerase amplification reactions using body heat, PLOS ONE, 9 (11) e112146. DOI: http://dx.doi.org/10.1371/journal.pone.0112146

The following two tabs change content below.
Avatar

Sara Klink

Technical Writer at Promega Corporation
Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

Leave a Reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.