PCR and qPCR
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Developing onsite genotyping of Antarctic penguins

Clade-specific PrimeTime® Custom qPCR Assays

Adélie Penguins

Adélie penguins live on sea ice but breed on ice-free land in Antarctica.

The challenges of developing a conservation program for Adélie penguins

Adélie penguins live only along the coastline of Antarctica and are considered “Near Threatened” [1], in part, because their sea-ice habitat is vulnerable to changes from global warming. Two genetic variants, or clades, of Adélie penguins have been identified. Understanding population trends across these clades will aid the design of conservation programs for these penguins.

Dr Jonathan Banks, Senior Scientist at the Cawthron Institute (see the Researcher profile), is studying the mating behaviors of Adélie penguins that live around the Ross Sea, approximately 4000 km south of New Zealand. Dr Banks can use DNA samples from feathers to determine whether selected mating pairs of Ross Sea penguins are from one or both Adélie clades. This process is complicated as the mating season is short and usually one of the mating pair feeds at sea while the other incubates the pair’s eggs on land. Having access to onsite molecular testing would simplify sample processing and ultimately increase the number of mating pairs that could be included in this project.

Selecting robust qPCR assays for a mobile PCR thermal cycler

In collaboration with Dr Jo-Ann Stanton (University of Otago; Otago, New Zealand) and Dr Elisabeth Wagner (IDT; Coralville, IA, USA), Dr Banks has validated qPCR assays that distinguish the two Adélie penguin clades [2]. By comparing genetic sequences from the two penguin types, Dr Wagner helped identify potential primer sites targeting the penguin mitochondrial genome. They identified a 138 nucleotide sequence within the d-loop region that varied between the two Adélie penguin groups and designed qPCR primers specific for each clade. Before use in the field, Drs Stanton and Banks validated the rigor of the SYBR® Green–based assays using archived penguin DNA stored under variable conditions. High resolution melt curves distinguished true positive results from amplification of primer-dimers. Clade identification results using the selected PrimeTime qPCR Assays completely corroborated data obtained by DNA sequencing. Complete details about this research and the assays are published in the New Zealand Journal of Zoology.

As a finalist of the Freedom for You grant sponsored by Ubiquitome, Dr Banks wants to use the Ubiquitome Freedom4 device (see the sidebar, From benchtop to handheld, battery-operated instrument) with the PrimeTime® qPCR Assays to perform onsite molecular genotyping in Antarctica. “Significant time and resources can be saved by eliminating the long, complex transport chain. Helicopters, light aircraft, and intercontinental aircraft are all required just to get samples to the New Zealand laboratory, and then return results to me in Antarctica,” notes Dr Banks.

Field testing around the world

Several researchers around the world wish to incorporate the Freedom4 device into field testing projects. To learn more about Dr Banks’ research and additional field application stories that use the Freedom4 device, visit www.UbiquitomeBio.com.

To read about the successful use of PrimeTime qPCR Assays with different species, instruments, and probe and intercalating dyes, see the list of peer-reviewed research articles on the PrimeTime citations page. The Related reading section below provides qPCR education and support materials for both novice and experienced qPCR researchers.

  1. BirdLife International 2012. Pygoscelis adeliae. The IUCN Red List of Threatened Species 2012: e.T22697758A40175259. www.iucnredlist.org (Accessed November 18, 2015).
  2. Banks JC, Clark JA, et al. (2016) Haplotyping cryptic Adélie penguin taxa using low-cost, high-resolution melt curves. New Zealand J Zool DOI: 10.1080/03014223.2015.1129971

Author: Maureen Young, PhD, is a Senior Scientific Writer at IDT.

Dr Jonathan Banks

Dr Jonathan Banks in Antarctica with emperor penguins.

Researcher profile

Dr Jonathan Banks, Senior Scientist, Cawthron Institute

Dr Jonathan Banks originally trained as a pharmacist. However, looking for new challenges, he began to study birds and obtained a master’s degree in behavioral ecology from Lincoln University (Lincoln, New Zealand). Then, with the support of a Kelly Tarlton’s Antarctica New Zealand scholarship, Dr Banks earned a PhD (Lincoln University) investigating co-evolution between lice and their penguin hosts in the Ross Sea, South America, the Falkland Islands, and South Africa. Subsequent postdoctoral training took him to the University of Illinois (Urbana Champaign, IL, USA), where he studied parasitic wasps and their commensal viruses. A FRST postdoctoral fellowship returned Dr Banks to New Zealand (University of Waikato; Hamilton, New Zealand) for a project using genetic fingerprinting to characterize the fecal flora of Adélie and emperor penguins, South Polar skuas, and Weddell seals. Catching 40 kg emperor penguins and 600 kg seals was certainly a different set of challenges from pharmacy!

Dr Banks is now a senior scientist at the Cawthron Institute (Nelson, New Zealand), an independent science organization that offers services related to aquaculture research, marine and freshwater resource management, food safety and quality, algal technologies, biosecurity, and analytical testing. His current research interests include microbial-based fecal source tracking and freshwater ecology, with a focus on penguin parasites and taxonomy.

The Freedom4 Device

The Freedom4 device.

From benchtop to handheld, battery-operated instrument

PCR experiments have quickly improved our understanding of basic biology and, increasingly, provide rapid, accurate testing in the applied sciences. Along with the development of applications, there have been refinements to the polymerases, primer designs, and thermal cycling machines. The next big step in the evolution of PCR thermal cyclers—a handheld, battery-operated qPCR instrument called Freedom4—was announced by Ubiquitome Limited in September 2014.

The instrument processes 4 reactions (sample plus negative, positive, and extraction controls) and is compatible with assays that use either SYBR® Green or FAM fluorescent dyes (used in intercalating dye assays and 5′ nuclease probe-based assays, respectively).

Product focus

PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies
  • Primer-based assays—designed for intercalating dye experiments

Design custom assays using our proprietary bioinformatics algorithms, any sequence, and your specific parameters. Alternatively, use predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime. Assistance with assay design is available through our Scientific Application Specialist team at applicationsupport@idtdna.com.

Related reading

Developing a qPCR point-of-care diagnostic for Ebola: Learn how Ubiquitome, Battelle, and IDT are developing a rapid qPCR Ebola virus test for easy use in the field. The PrimeTime qPCR assay is designed to be run on Ubiquitome’s hand-held, battery powered real-time PCR device, the Freedom4.

DECODED 4.2—special qPCR issue: Get this compendium of our newsletter articles about qPCR in one convenient volume.

PrimeTime® qPCR Application Guide: Download a useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting.

© 2015 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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