Improving DNA amplification from problematic plants

Jan 03, 2013

The polymerase chain reaction (PCR) is a common technique used to amplify, or copy, pieces of DNA. Amplified DNA is then used in genetic analyses for everything from medicine to forensics. In plant research, PCR is a vital step in detecting and sequencing genes, and its applications are endless. However, compounds found in plants often inhibit PCR. Researchers at the University of Southern Mississippi discovered that the use of an additive allows PCR to successfully amplify DNA from once problematic plants.

PCR is widely used in plant sciences but is not 100 percent reliable. Many plant researchers encounter roadblocks when implementing PCR. For example, many plant species contain that deter herbivores. These compounds are often extracted along with and can stop PCR from working.

Graduate student Tharangamala Samarakoon and colleagues have found a technique to overcome many of these inhibitory . They added a reagent to the PCR mixture that contains three ingredients: trehalose, , and polysorbate-20 (all three abbreviated TBT-PAR). "Unlike several other studies, TBT-PAR works at the PCR stage instead of at the DNA extraction stage, so it has promise for pigeon-holed and half-forgotten extractions that previously failed to be amplified using PCR," says Samarakoon. The authors published their research in the January issue of Applications in Plant Sciences.

Samarakoon tested the TBT-PAR reagent on DNA extracted from tropical and temperate species across four plant families, including Achariaceae, Asteraceae, Lacistemataceae, and Samydaceae. PCR with TBT-PAR successfully amplified DNA for all species, whereas standard DNA extraction and PCR techniques consistently failed.

TBT-PAR enhanced PCR for DNA extracted from fresh, silica-dried, and herbarium plant material. "Since we study , many of which are geographically restricted or rare," explains Samarakoon, "herbarium material is sometimes all that we have available for , and curators are gracious to allow even a small destructive sampling for a single extraction attempt. We want that one attempt, of course, to be successful." Samarakoon predicts that inhibitory plant compounds could be the underlying cause of many PCR failures in herbarium specimens and hopes TBT-PAR will have widespread benefits in herbarium specimen DNA amplification.

TBT-PAR was first used in the PCR detection of a shrimp virus by co-author Shiao Wang and his colleagues. "The additive has also been helpful in a colleague's lab where they had trouble amplifying DNA from gopher tortoise ticks, so its utility extends beyond plants," comments Samarakoon. TBT-PAR has the potential for broad use in PCR techniques across DNA samples, species, and taxa.

The article will be published in the first issue of Applications in Plant Sciences (APPS), a new journal released by the Botanical Society of America. Theresa Culley, Editor-in-Chief of APPS, describes the new journal as a venue to "expedite the dissemination of innovative information encompassing all areas of the plant sciences, including but not limited to genetics, structure, development, evolution, systematics, and ecology." APPS publishes new methods in plant sciences—an important niche to fill in an age of rapid technological advances.

Explore further: First sex determining genes appeared in mammals 180 million years ago

More information: Culley, T. M. 2013. Changing technologies offer new opportunities in the plant sciences. Applications in Plant Sciences 1(1): 1200008. doi:10.3732/apps.1200008

Samarakoon, T., S. Y. Wang, and M. H. Alford. 2013. Enhancing PCR amplification of DNA from recalcitrant plant specimens using a trehalose-based additive. Applications in Plant Sciences 1(1): 1200236. doi:10.3732/apps.1200236

add to favorites email to friend print save as pdf

Related Stories

New DNA analysis thousand times more sensitive

Jun 17, 2011

(PhysOrg.com) -- An international team of researchers has developed a new DNA technology which makes it possible to perform reliable analyses on DNA quantities that are a thousand times smaller than was previously the case. ...

Recommended for you

Genetic code of the deadly tsetse fly unraveled

15 hours ago

Mining the genome of the disease-transmitting tsetse fly, researchers have revealed the genetic adaptions that allow it to have such unique biology and transmit disease to both humans and animals.

Engineered E. coli produces high levels of D-ribose

16 hours ago

D-ribose is a commercially important sugar used as a sweetener, a nutritional supplement, and as a starting compound for synthesizing riboflavin and several antiviral drugs. Genetic engineering of Escherichia co ...

User comments : 0

More news stories

Genetic legacy of rare dwarf trees is widespread

Researchers from Queen Mary University of London have found genetic evidence that one of Britain's native tree species, the dwarf birch found in the Scottish Highlands, was once common in England.

Ocean microbes display remarkable genetic diversity

The smallest, most abundant marine microbe, Prochlorococcus, is a photosynthetic bacteria species essential to the marine ecosystem. An estimated billion billion billion of the single-cell creatures live i ...

Genetic code of the deadly tsetse fly unraveled

Mining the genome of the disease-transmitting tsetse fly, researchers have revealed the genetic adaptions that allow it to have such unique biology and transmit disease to both humans and animals.

Cell resiliency surprises scientists

New research shows that cells are more resilient in taking care of their DNA than scientists originally thought. Even when missing critical components, cells can adapt and make copies of their DNA in an alternative ...

Google+ boss leaving the company

The executive credited with bringing the Google+ social network to life is leaving the Internet colossus after playing a key role there for nearly eight years.