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PCR Enhancement Additive Improves DNA Amplification from Recalcitrant Plant Species

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Biologists from the University of Southern Mississippi have concocted a chemical additive that enhances PCR amplification of several recalcitrant plant specimens in which conventional PCR is unsuccessful, according to recently published research.

According to the researchers, the additive is inexpensive and easy to make, and works well not only with DNA from plant samples that have proven particularly difficult in the past, but with samples that have generally not been problematic for PCR. As such, it could be useful as a broad addition to any PCR amplification of plant samples.

In addition, they have distributed their additive to other laboratories, which have used it to successfully PCR amplify nucleic acids from known recalcitrant samples such as insects and fish, which suggests that the chemical mixture may even be broadly useful outside of plant biology.

The researchers, from USM's Department of Biological Sciences, described their additive in a paper published this month in the inaugural online edition of Applications in Plant Science, a publication of the Botanical Society of America.

Tharangamala Samarakoon, a graduate student in the department and first author on the paper, told PCR Insider this week that the problem of plant compounds such as polysaccharides and phenolic compounds inhibiting DNA amplification is widespread. "Since negative results are not always published, the issue is [more] widespread than we think," Samarakoon said.

Samarakoon became involved in the project while she was conducting research on plant species from tropical countries. Many of these species, she said, contain multiple PCR inhibitors, "so I couldn't get positive results from PCR. I worked for two or three years … to get good sequences for my plant, which is why I looked for an alternate way to get [PCR] results." In many cases, failure to amplify DNA from particular plant species persisted even when template DNA was extracted from fresh tissue and was of high yield and quality.

Her USM colleague and a co-author on the paper, Shiao Wang, had previously been successful using an additive based on a disaccharide called trelahose to successfully PCR amplify DNA from shrimp viruses, and he suggested that Samarakoon try the same method in her plant studies.

After tinkering with the formula, the researchers wound up with a reagent containing trehalose, bovine serum albumin, and polysorbate-20, a surfactant also known as Tween 20. According to the researchers, trehalose is known to stabilize proteins and backbone fluctuations of DNA. Meantime, BSA, which is a common additive to PCR solutions, is known to have high lysine content, and phenolic compounds may bind with lysine and thereby prevent their binding to and inactivation of Taq polymerase.

Finally, the addition of nonionic detergents like Tween-20 can neutralize the negative effects of sodium dodecyl sulfate. According to the researchers, as little as 0.01 percent contamination of template DNA leftover from extraction can inhibit PCR by reducing Taq polymerase activity to as low as 10 percent.

Using their additive, called TBT-PAR, the researchers tested template DNA from various species of the Achariaceae, Asteraceae, Lacistemataceae, and Samydaceae families. The DNA was extracted from fresh samples, field samples collected in silica gel, and old herbarium specimens that had previously never produced positive PCR results with standard techniques.

They compared PCR amplification of these samples both with and without the TBT-PAR additive, and found that the enhancer consistently improved the efficacy of PCR amplification. Among DNA samples extracted from fresh or silica-dried leaves from eight recalcitrant species of Samydaceae, one — a control sample — was PCR amplified successfully in the absence of TBT-PAR, but all amplified with the addition of TBT-PAR.

Similarly, among DNA extracted from herbarium specimens of eight species, one — again, a control known to amplify under standard conditions — amplified successfully without TBT-PAR, but all amplified with the addition of TBT-PAR.

The researchers did note slight differences in the sizes of PCR products from both methods, chalking this up to variable-length repeats within the trnL-F spacer, which often differ in length even in closely related species.

In addition, "PCR inhibition was probably not eliminated completely as evidenced by the varying yields. Other possible causes for the difference in PCR yield among samples include low template DNA concentration and/or possible partial DNA degradation," the researchers wrote.

Still, they noted that all TBT-PAR amplifications of fresh or silica-dried material have yielded positive results so far, and about 80 percent of trials with herbarium material dating to 1989 have yielded positive results.

Samarakoon said that her lab has shared its additive with other USM researchers that were having problems PCR amplifying recalcitrant samples from animals — for instance gopher tortoise ticks and certain fish — and "all have gotten good results."

Samarakoon's colleague Wang noted in an email that he has also send a sample of the additive to research groups in Florida and Texas, who have reported similar success. "I think both worked on plants. Within the university here, I’ve given the additive to several graduate students who had trouble amplifying their DNA of interest."

Sample types have included plants, viruses, blood, and solid tissue, Wang noted, although he couldn't provide further detail, noting that he hasn't really kept track of anecdotal results.

"Basically, they mention trouble with PCR, I give them the additive to try, and that’s pretty much it," he said, adding that responses have generally been "uniformly positive."

These successes, the researchers noted, mean that their additive may have broad use outside of plant biology for enhancing PCR amplification of recalcitrant species. The researchers haven't tried using TBT-PAR with human samples yet, but it is commonly known that human stool samples are notorious for containing PCR inhibitors — especially complex polysaccharides — that can confound results.

The USM researchers have not applied for patents on their work. Samarakoon said that the additive is "based on very simple ingredients, very cheap and easy. I think anybody can make their own."

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