Tips and Tools

Elevate Your Research: Exploring the Power of 8-Dye STR Chemistry with the Spectrum Compact CE System

Posted on by Promega

In genetic research, staying at the forefront of technology is crucial. The latest breakthrough in human identification comes in the form of 8-dye Short Tandem Repeat (STR) chemistry. This innovation promises unprecedented precision and accuracy in DNA analysis, revolutionizing the way we approach genetic studies. In this blog post, we’ll delve into the world of 8-color chemistry and explore how it seamlessly integrates with the game-changing Spectrum Compact CE System.

Understanding 8-Dye STR Chemistry

The introduction of 8-dye chemistry expands the capability of STR analysis, enabling researchers to analyze more DNA markers with smaller amplicons, providing more robust data from degraded or inhibited DNA samples.  The performance of the 8-color dye chemistries from Promega on the Spectrum Compact CE System is sensitive, with both chemsitries (PowerPlex® 35 GY System and the upcoming PowerPlex® 18 E System) producing 100% profiles from their suggested inputs down to as little as 62.5 pg of DNA. The 18E system produced 100% profiles down to 31.25 pg of input DNA with minimal signal bleed through and low system noise.

Table showing percent STR profiles generated with decreasing input DNA using the PowerPlex 35GY or PowerPlex 18 E chemistry on the Spectrum Compact CE System
Table showing percent profiles generated with decreasing input DNA using the PowerPlex® 35GY or PowerPlex® 18E chemistry on the Spectrum Compact CE System.
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Promega qPCR Grant Series #2: Molecular Biologist, Laura Leighton

Posted on by Anna Bennett

Our second installment of the Promega qPCR Grant Recipient blog series highlights Dr. Laura Leighton, a trained molecular biologist and postdoctoral researcher at the Australian Institute for Bioengineering and Nanotechnology. Leighton’s scientific journey features a passion for molecular biology and problem-solving. Her path has been illuminated by mentorship, relationships with fellow scientists and a commitment to creativity in overcoming challenges. Here, we explore her scientific journey, reflect on research lessons and foreshadow her plans for the Promega qPCR grant funds.

Dr. Laura Leighton grew up in a rural area in Far North Queensland, Australia, where she spent her early life exploring critters on the family farm. Her upbringing was infused with a deep connection to the environment, from raising tadpoles in wading pools to observing wildlife and witnessing food grow firsthand. Observing the biology around her ultimately piqued her interest in science from a young age. She then began her academic journey in 2011 at the University of Queensland, Australia. She studied biology while participating in a program for future researchers, which led her to undergraduate research work in several research labs.  She dabbled in many research avenues in order to narrow in on her scientific interests all while adding different research tools to her repertoire.

After serving as a research assistant in Dr. Timothy Bredy’s lab, she decided to continue work in this lab and pursue a PhD in molecular biology. During her PhD, Leighton worked on several projects from cephalopod mRNA interference to neurological wiring in mice. The common thread in these projects is Leighton’s passion for the puzzles of molecular biology:

“I also love molecular engineering and the modularity of molecular parts. There’s something really special about stringing together sequence in a DNA editor, then seeing it come to life in a cell,” she says.

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RAF Inhibitors: Quantifying Drug-Target Occupancy at Active RAS-RAF Complexes in Live Cells

Posted on by Ken Doyle

Mitogen-activated protein kinases (MAPKs) are a large family of proteins that regulate diverse cellular functions in eukaryotes, including gene expression, proliferation, differentiation and apoptosis (1). MAPK signaling pathways typically include three sequentially activated kinases, and these pathways are triggered in response to extracellular stimuli, such as cytokines, mitogens, growth factors and oxidative stress (1). Ultimately, the signal is transmitted to the nucleus, with the activation of a specific transcription factor that modulates the expression of one or more genes.

Among MAPK pathways, the RAS-RAF-MEK-ERK signaling pathway has been studied extensively. Mutations in RAS family proteins and resultant dysregulation of the signaling pathway are implicated in a variety of cancers. Therefore, this pathway is a popular target for anticancer drug development.

An overview of the RAS-RAF-MEK-ERK signaling pathway.
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Promega qPCR Grant Series #1: Marine Plant Ecologist, Dr. Agustín Moreira-Saporiti

Posted on by Anna Bennett
Dr. Agustín Moreira-Saporiti is a postdoctoral researcher at the Marine Biological Laboratory and is studying flowering processes in marine seagrass

Marine seagrasses are submerged flowering plants that form essential underwater meadows, fostering diverse ecosystems and providing a habitat for marine life. Our first Promega qPCR Grant winner and marine ecologist, Dr. Agustín Moreira-Saporiti, plans to continue adding to a fascinating body of work aimed at understanding flowering in marine seagrasses.

Dr. Moreira-Saporiti began his journey into marine plant ecology at the University of Vigo, Spain, where he earned a bachelor’s degree in marine sciences. He then went on to complete a master’s degree at the University of Bremen (Germany) where his thesis focused the ecology of seagrasses in Zanzibar, Tanzania. His passion for marine botany led him down a deeper exploration of marine plants, unraveling the intricate web of ecosystem processes within seagrasses.

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On-site, In-house Environmental Monitoring to Obtain Species-Level Microbial Identification

Posted on by Michele Arduengo

Loss of life and serious illness from contamination of manufactured products that are consumed as food or used in medical procedures illustrate the need to prevent contamination events rather than merely detect them after the fact.  High-profile news stories have described contamination events in compounding pharmacies (1), food processing and packaging plants (2) and medical device manufacturers (3). Although contamination in manufacturing settings can be physical, chemical, or biological, this article will focus environmental monitoring to determine the quality of a manufacturing facility with respect to microbial contamination.

Scientist in pharmaceutical manufacturing facility performing environmental monitoring.

To ensure that the products they produce and package are manufactured in a high-quality, contaminant-free environment, many industries are required to establish routine environmental monitoring programs. Samples are collected from all potential sources of contamination in the production environment including air, surfaces, water supplies and people. Routine monitoring is essential to detect trends such as increases in potential pathogens over time or the appearance of new species that have not been seen before so that contamination events can be prevented.

Because environmental monitoring requires identification to the level of the species, most environmental monitoring programs will collect samples and then send them off to a facility to be sequenced for genomic identification of any microbial species. Such genotypic analysis involves DNA sequencing of ribosomal RNA (rRNA) genes to determine the taxonomic classification of bacteria and fungi. In this method, informative sections of the rRNA genes are amplified by PCR; the PCR products sequenced; the sequence is compared to reference libraries; and the results interpreted to make a species-level identification for a given microbial isolate.

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From Hit to Live-Cell Target Engagement Assay: DNA-Encoded Library Screens and NanoBRET™ Dye

Posted on by Tian Yang

Monitoring and quantifying drug-target binding in a live-cell setting is important to bridging the gap between in vitro assay results and the phenotypic outcome, and therefore represents a crucial step in target validation and drug development (1). The NanoBRET™ Target Engagement (TE) assay is a biophysical technique that enables quantitative assessment of small molecule-target protein binding in live cells. This live-cell target engagement assay uses the bioluminescence resonance energy transfer (BRET) from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent tracer that reversibly binds the target protein of interest. In the presence of unlabeled test compound that engages the target protein, the tracer is displaced, and a loss of BRET signal is observed. Due to the tight distance constraints for BRET, the signal measured is specific to the target fused to NanoLuc® luciferase.

Live-cell target engagement assay using NanoBRET to measure small molecule binding to a target transmembrane protein.

Promega offers over 400 ready-to-use assays for multiple target classes, including kinases, E3 ligases, RAS, and many others. For targets that do not have an existing NanoBRET™ TE assay, Promega offers NanoBRET™ dyes, NanoLuc® cloning vectors, and NanoBRET™ detection reagents to develop novel NanoBRET™ TE assays.

To learn more about the NanoBRET™ TE platform, see the NanoBRET™ Target Engagement Technology Page on our website.

One critical component in the development of novel NanoBRET™ TE assay is the creation of the cell-permeable fluorescent tracers (NanoBRET™ tracers) against the target protein of interest. The tracers are bifunctional, consisting of a NanoBRET™-compatible fluorophore and a target-binding moiety connected by a linker. While the NanoBRET™ 590 dyes have demonstrated consistently robust cell permeability and optimal spectral overlap with NanoLuc® for BRET, a ligand capable of binding to the target protein of interest needs to be identified to generate a NanoBRET™ tracer.

What Are DNA-Encoded Libraries?

DNA-Encoded Libraries, (DELs), have emerged as powerful tools for discovering small molecule ligands to target proteins of interest at an unprecedented scale. . owing to the ability of a DEL  to enable the synthesis of larger libraries of compounds and to target proteins without any prior structural knowledge of the proteins or their ligands (2). Because each member of a DEL contains a DNA barcode and a small molecule separated by a linker, DEL is primed for discovering leads within therapeutic modalities that rely on bifunctional chemistry, such as proteolysis targeting chimeras (PROTACs). Since NanoBRET™ tracers are also bifunctional, ligands identified from DEL selections could serve as ideal candidates for developing novel NanoBRET™ tracers that can enable NanoBRET™ TE assays for new targets.

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Insects and Science: Optimizing Work with Sf9 Insect Cells

Posted on by Shannon Sindermann

Insects are a keystone species in the animal kingdom, often providing invaluable benefits to terrestrial ecosystems and useful services to mankind. While many of them are seen as pests (think mosquitos), others are important for pollination, waste management, and even scientific research.

Insect biotechnology, or the use of insect-derived molecules and cells to develop products, is applied in a diverse set of scientific fields including agricultural, industrial, and medical biotechnology. Insect cells have been central to many scientific advances, being utilized in recombinant protein, baculovirus, and vaccine and viral pesticide production, among other applications (5).

Therefore, as the use of insect cells becomes more widespread, understanding how they are produced, their research applications, and the scientific products that can be used with them is crucial to fostering further scientific advancements.

Primary Cell Cultures and Cell Lines

Cell culture - Cell lines - Insect Cells

In general, experimentation with individual cells, rather than full animal models, is advantageous due to improved reproducibility, decreased space requirements, less ethical concerns, and a reduction in expense. This makes primary cell cultures and cell lines essential contributors to basic scientific research.

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No Horsin’ around with Halal Meat Authentication

Posted on by Promega

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.  

People’s diets are frequently influenced by a wide range of variables; with environment, socioeconomic status, religion, and culture being a few of the key influencers. The Muslim community serves as one illustration of how culture and religion can hold influence over people’s eating habits.

Halal meat on cutting board

Muslims, who adhere to Islamic teachings derived from the Qur’an, frequently base dietary choices on a food’s halal status, whether it is permissible to consume, or haram status, forbidden to consume. With the population of Muslims expected to expand from 1.6 billion in 2010 to 2.2 billion by 2030, the demand for halal products is anticipated to surge (2).

By 2030, the global halal meat market is projected to reach over $300 billion dollars, with Asia-Pacific and the Middle East regions being the largest consumers and producers of halal meat products (3). Furthermore, increasing awareness and popularity of halal meat among non-Muslim consumers, as well as strengthening preference for ethical and high-quality meat, are all contributing to demand.  

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Streamlining Disease Diagnostics to Protect Potato Crops

Posted on by AnnaKay Kruger
A potato farmer holds a handful of potatoes. Scientists are working to protect potato crops from disease.
The WSPCP works to provide seed potato growers with healthy planting stock

The mighty potato—the Midwest’s root vegetable of choice—is susceptible to a variety of diseases that, without proper safeguards, can spell doom for your favorite side dishes. Founded in 1913 and housed in the Department of Plant Pathology at the University of Wisconsin-Madison, the Wisconsin Seed Potato Certification Program (WSPCP) helps Wisconsin seed potato growers maintain healthy, profitable potato crops year-to-year through routine field inspections, a post-harvest grow-out and laboratory testing.

While WSPCP conducts visual inspections for various seed potato pathogens, their diagnostic laboratory testing is primarily focused on viruses such as Potato virus Y (PVY), which can cause yield reduction and tuber defects, along with select bacteria such as Dickeya and Pectobacterium species that cause symptoms like wilting, stem rot and tuber decay.

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Custom Manufacturing: Translating Research into Product

Posted on by Natalie Larsen

Scientists around the world are focusing their energy and resources on translating advances made in clinical research into relevant biotechnology, clinical, and applied products that improve our health and well-being. Once research looks promising, there is substantial pressure to expedite the release of that product or assay in the market.

For many organizations focused on developing these advanced products, their expertise and core competencies are in developing the assay. Often, they do not have the experience, infrastructure, or quality systems in place to support large-scale production, packaging, or distribution of their newly developed assay in a way that is also in compliance with relevant regulatory requirements. These next steps become a barrier to realizing the value of the research. Working with a custom or contract manufacturing partner can lower this barrier and expedite the time to market.

Custom Manufacturing

Be careful not to confuse custom manufacturing with original equipment manufacturer (OEM) products. OEM products are existing products from one company that another company rebrands and sells. Custom manufacturers typically focus on providing more comprehensive services that can be adapted to produce a new product. Custom manufacturing is not “one size fits all” and can be simple or complex, such as producing a single component to a final finished product.

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