Endpoint PCR in 15 Minutes with New Master Mix and Thermal Cycler Combination

Since its invention in 1983, the polymerase chain reaction (PCR) has become a fundamental technology in life science laboratories across the world. Much of the technological innovation is driven by quantitative PCR and digital PCR (1); however, endpoint PCR remains a workhorse technology for applications such as gene cloning, mutagenesis and detection of microbial pathogens. Variations on the basic endpoint PCR method—for example, the use of multiplexed, fluorescently labeled primers followed by capillary electrophoresis to analyze the amplified DNA fragments—are popular in forensic DNA analysis and cell line authentication.

The COVID-19 pandemic has created an urgent need for PCR-based diagnostic testing for SARS-CoV-2. Most of these diagnostic tests use real-time, reverse-transcription quantitative PCR (RT-qPCR). However, RT-qPCR can be challenging for routine use in developing countries and in laboratories with limited access to real-time PCR thermal cyclers. A recent study described an endpoint PCR method for SARS-CoV-2 detection to address these limitations (2).

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RNA-Protein Interactions: A New Frontier for Drug Discovery

Almost 90% of the human genome is transcribed into RNA, but only 3% is ultimately translated into a protein. Some non-translated RNA is thought to be useless, while some play a significant yet often mysterious role in cancer and other diseases. Despite its abundance and biological significance, RNA is rarely the target of therapeutics.

“We say it’s undruggable, but I would say that ‘not-yet-drugged’ is a better way to put it,” says Amanda Garner, Associate Professor of Medicinal Chemistry at the University of Michigan. “We know that RNA biology is important, but we don’t yet know how to target it.”

Amanda’s lab develops systems to study RNA biology. She employs a variety of approaches to analyze the functions of different RNAs and study their interactions with proteins. Her lab recently published a paper describing a novel method for studying RNA-protein interactions (RPI) in live cells. Amanda says that with the right tools, RPI could become a critical target for drug discovery.

“It’s amazing that current drugs ever work, because they’re all based on really old approaches,” Amanda says. “This isn’t going to be like developing a small molecule kinase inhibitor. It’s a whole new world.”

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Galloping to Greatness: Meet Kurt the First Cloned Przewalski’s Horse

Kurt the first cloned Przewalsk'si horse
Kurt the foal at the Texas veterinary facility of ViaGen Equine collaborator, Timber Creek Veterinary, August 28, 2020.
Photo provided courtesy of Scott Stine.

On August 6, 2020, the first successfully cloned Przewalski’s horse was born at the Texas-based veterinary facility, Timber Creek Veterinary, along with a new hope for restoring some much-needed genetic diversity to the species. The successful birth of this foal is the culmination of the collaborative efforts between Revive & Restore, San Diego Zoo Global (SDZG), and ViaGen Equine, and lays the groundwork as an important model for future conservation efforts.

The new Przewalski’s foal (pronounced “shuh-VAL-skees”) has been affectionately dubbed Kurt, in honor of noted animal conservationist, geneticist and pathologist, Dr. Kurt Benirschke. Dr. Benirschke played an instrumental role in founding the Frozen Zoo®, a genetic library comprised of cryopreserved cell lines of endangered species. Established in the 1970s, this collection was built on a foundation of prescient hope, banking on the future development of reproductive and cloning technologies that did not yet exist.

Now thanks to his foresight, that gamble is paying off and the fruits of that labor are literally being brought to life almost 50 years later through Kurt the foal, who is as adorable as he is important to the future of his kind.

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Identifying the Ancestor of a Domesticated Animal Using Whole-Genome Sequencing

What animal can be found around the globe that outnumbers humans three to one? Gallus gallus domesticus, the humble chicken. The human appetite for eggs and lean meat drive demand for this feathered bird, resulting in a poultry population of over 20 billion.

Controversy over the origin of the domestic chicken (when, where and which species) have lead some researchers to look for that information in the genomes of contemporary chicken breeds and wild jungle fowl, the candidates from which chickens were derived. By sequencing over 600 genomes from Asian domestic poultry as well as 160 genomes from all four wild jungle fowl species and the five red jungle fowl subspecies, Wang et al. wanted to understand and identify the relationships and relatedness among these species and derive where domesticated chickens first arose.

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XpressAmp™ Direct Amplification: Simplified and Accelerated Time to qPCR Results

As the SARS‐CoV‐2 pandemic continues to rage across the United States and around the globe, the demand for COVID‐19 testing is increasing. The vast majority of the COVID-19 assays use RT‐qPCR to detect the viral RNA in patient samples such as nasopharyngeal swabs, which are collected and stored in viral or universal transport media (VTM/UTM). The general workflow for these COVID‐19 assays can be broken down as follows:

  1. Collect and store patient samples
  2. Ship samples to testing laboratory
  3. Extract RNA from samples
  4. Amplify and analyze samples

While many companies who manufacture the products that are used in these steps have been able to adapt and significantly increase their production capacities, there are still gaps between the supply of these products and the global test demand. Both the sample collection and storage step and the RNA extraction/purification step have a tendency to bottleneck and experience supply constraints. One way to address these bottlenecks and expand production capacity for these in‐demand products is to evaluate the viability of skipping a step in the workflow, without hindering the ability to detect viral RNA from samples.

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Capillary Electrophoresis On Your Benchtop

Spectrum Compact CE System

Here’s the good news: The Spectrum Compact CE System is now available from Promega. 

Here’s the better news: Labs of all sizes now have the opportunity to perform Sanger sequencing and fragment analysis with a personal, benchtop instrument. 

There is no bad news. 

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Lessons in History, Hope and Living with Lynch Syndrome from the “Daughter of Family G”

Lynch Syndrome is the autosomal dominant hereditary predisposition to develop colorectal cancer and certain other cancers. This simple, one sentence definition seems woefully inadequate considering the human toll this condition has inflicted on the families that have it in their genetic pedigree.

They Called it a Curse

To one family, perhaps the family when it comes to this condition, Lynch Syndrome has meant heartache and hope; grief and joy; death and life. Their story is told by Ami McKay in her book Daughter of Family G, and it is at once both a memoir of a Lynch Syndrome previvor (someone with a Lynch Syndrome genomic mutation who has not yet developed cancer) and a poignant and honest account of the family that helped science put name to a curse.

“The doctors called it cancer. I say it’s a curse. I wish I knew what we did to deserve it.”

Anna Haab from Daughter of Family G (1)

The scientific community first met “Family G” as the meticulously created family tree, filled with the stunted branches that mark early deaths by cancer. The pedigree was first published in 1913 in Archives of Internal Medicine (2). In the article, Dr. Alderd Warthin wrote: “A marked susceptibility to carcinoma exists in the case of certain family generations and family groups.” In 1925, an expanded pedigree of circles and squares was published in Dr. Warthin’s follow up study in the Journal of Cancer Research (3).  But each circle and square in that pedigree denotes a person. Each line represents their dreams together for the future, and Ms. McKay wants us to know their names: Johannes and Anna, Kathrina, Elmer, Tillie, Sarah Anne (Sally); and—most importantly—Pauline. Because without Pauline there would be no story.

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What We Know About the COVID-19 and the SARS-CoV-2 Virus

David Goodsell image of SARS-2-CoV
Image by David Goodsell

In the nine months since the first cases of COVID-19 were noticed in Wuhan, China, the virus has spread around the globe and infected over 22 million people. As with all emerging infectious diseases, we often find ourselves with more questions than answers. However, through the tireless work of researchers, doctors and public health officials worldwide, we have learned a lot about the virus, how it spreads and how to contain it.

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Using the Power of Technology for Viral Outbreaks

Artist’s rendition of a virus particle.

When the world is experiencing a viral pandemic, scientists and health officials quickly want data-driven answers to understand the situation and better formulate a public health response. Technology provides tools that researchers can use to develop a rapid sequencing protocol. With such a protocol, the data generated can help answer questions about disease epidemiology and understand the interaction between host and virus. Even better: If the protocol is freely available and based on cheap, mobile sequencing systems.

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Emergency Use Authorization: The What, the Why and the How

This blog is written by guest blogger, Heather Tomlinson, Director of Clinical Diagnostics at Promega.

Finding safe and effective treatments for human diseases takes time.  Medication and diagnostic tests can take decades to discover, develop and prove safe and effective.  In the United States, the FDA stands as the gold-standard gatekeeper to ensure that treatments and tests are reliable and safe. The time we wait in review and clearance means less risk of ineffective or unsafe treatments.

And yet, in a pandemic, we are behind before we even start the race to develop diagnostic tests, so critical for understanding how an infectious disease is spreading. That is when processes like the FDA’s fast track of Emergency Use Authorization (EUA) are critical. Such authorization allows scientists and clinicians to be nimble and provide the best possible test protocol as quickly as possible, with the understanding that these protocols will continue to be evaluated and improved as new information becomes available. The EUA focuses resources and accelerates reviews that keep science at the fore and gets us our best chance at staying safe and healing.

The Maxwell 48 RSC Instrument and the Maxwell RSC Total Viral Nucleic Acid Isolation Kit are now listed as options within the CDC EUA protocol.

For scientists working around the clock, the FDA’s EUA process is ready to review and respond. Getting an EUA  gives clinical labs a very specific and tested resource to guide them to the tools and tests to use in a crisis.

Typically the Centers for Disease Control (CDC) will develop the first test or protocol that receives FDA EUA in response to a crisis like a pandemic.  For COVID-19 the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel received FDA EUA clearance in early February. This is the test protocol used by the public health labs that work with the CDC and test manufacturers around the world.

Throughout a crisis such as the current pandemic, scientists continually work to improve the testing protocols and add options to the EUA protocols. This enables more flexibility in the test protocols. Promega is fortunate to play a part of the CDC EUA equation for diagnostic testing. Our GoTaq® Probe 1-Step PRT-qPCR System is one of a few approved options for master mixes in the  CDC qPCR diagnostic test,  and now our medium-throughput Maxwell 48 Instrument and Maxwell Viral Total Nucleic Acid Purification Kit  have been added to the CDC protocol as an option for the RNA isolation step as well. These additions to the CDC EUA means that laboratories have more resources at their disposal for the diagnostic testing which is so critical to effective pandemic response.

The Emergency Use Authorization provides the FDA guidance to strengthen our nation’s public health during emergencies, such as the current COVID-19 pandemic. The EUA allows continual improvement of an authorized protocol through the collaborative efforts scientists in all academia, government and industry to identify and qualify the most reliable technologies and systems, giving labs more flexibility as new products are added as options.

Dr. Tomlinson is the Director for the Global Clinical Diagnostics Strategic Business Unit at Promega Corporation with over 15 years of experience in clinical diagnostic test development. She is responsible for leading the team that drives strategy in the clinical market for Promega. Her background is in infectious disease diagnostic testing, with a focus on HIV drug resistance and evolution.  Her recent work has been in oncology companion diagnostic test development.  Heather has is an accomplished international presenter, delivering conference presentations in the United States, Europe, Asia, and Africa.

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