Nucleic Acid Analysis

Investigation of Remdesivir as a Possible Treatment for SARS-CoV-2 (2019-nCoV)

Posted on by Kyle Hooper

Remdesivir (RDV or GS-5734) was used in the treatment of the first case of the SARS-CoV-2 (formerly 2019-nCoV ) in the United States (1). RDV is not an approved drug in any country but has been requested by a number of agencies worldwide to help combat the SARS-CoV-2 virus (2). RDV is an adenine nucleotide monophosphate analog demonstrated to inhibit Ebola virus replication (3). RDV is bioactivated to the triphosphate form within cells and acts as an alternative substrate for the replication-necessary RNA dependent RNA polymerase (RdRp). Incorporation of the analog results in early termination of the primer extension product resulting in the inhibition.

Note the spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. In this view, the protein particles E, S, M, and HE, also located on the outer surface of the particle, have all been labeled as well. A novel coronavirus virus was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China in 2019.
This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM CDC

Why all the interest in RDV as a treatment for SARS-CoV-2 ? Much of the interest in RDV is due to a series of studies performed by collaborating groups at the University of North Carolina Chapel Hill (Ralph S. Baric’s lab) and Vanderbilit University Medical Center (Mark R. Denison’s lab) in collaboration with Gilead Sciences. 

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Researching the Researchers: Alberto Biscontin

Posted on by Promega

2019 Real-Time PCR Grant

The three 2019 Real-Time PCR Grant Winners have been hard at work in the six months since winning their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.

One of the 2019 winners, Alberto Biscontin (University of Padova, Italy), performs research in the fields of Neurogenetics and Chronobiology. He is looking to shed greater light on the circadian rhythms of the Antarctic krill. Alberto published his most recent analysis in Nature and GoTaq® qPCR Master Mix helped him validate expression of genes for his study.

His qPCR data showed support for internal mechanisms that not only support daily living but also clarified the overwintering process of the krill. Now that Alberto has sized up some zooplankton, we asked him to share a little more about himself and his research:

Q: How long have you been a researcher?
A: I have been a researcher since 2012.

Q: How did you decide to research Antarctic krill?
A: In 2013, I had the opportunity to join the international Antarctic research program PolarTime. [It] brought together eight research groups with different scientific expertise to study seasonal and daily rhythms in the Antarctic krill Euphausia superba.

Q: When you are not busy at the bench, what do you like to do?
A: Traveling. I love strolling through open-air markets.

Q: Are there any tips or tricks you have learned that make your job easier?
A: You can easily switch from a classic RT-PCR protocol to a cheaper and faster One-step protocol using the same primers and temperatures.

Q: What comes next?
A: I would like to characterize the clock machinery of other polar organisms to understand whether high latitude clocks have developed similar strategies to cope with [the] polar environment. Moreover, a better understanding of marine circadian clocks could help to shed light on the evolution of the animal circadian machinery.

You can find Alberto’s most recent publication in Nature Scientific Reports. The 2020 Real-Time PCR Grant will be coming soon. For more information on the 2019 winners and information on the 2023 Grant, visit the Real-Time Grant web page. Be sure to follow us on social media for the most up-to-date information regarding the 2020 Grant, including application deadlines and winner notifications!

We’re committed to supporting scientists who are using molecular biology to make a difference. Learn more about our qPCR Grant program.  

Related Posts

Cloning with pGEM®-T Vectors: Ligation

Posted on by Leah Cronan
T-Vector Cloning

One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems. This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The insert is directly ligated to the T-tailed plasmid vector with T4 DNA ligase. The insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the multiple cloning region of the T vector.

Proofreading polymerases like Pfu do not add “A” overhangs so PCR products generated with these polymerases are blunt-ended. In a previous blog, we discussed a simple method for adding an A-tail to any blunt-ended DNA fragment to enable T-vector cloning. Below, we think about the next step: Ligation.

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CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology

Posted on by Ken Doyle

Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.

Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.

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Minimizing Cross-Contamination Risk During Automated Processing of FFPE Tissues

Posted on by Eric Vincent

This is part 3 of a three-part series on FFPE sample processing. Part 1 (link) Part 2 (link)

I would like to automate FFPE processing, but I am worried about sample cross contamination, how can I minimize my risks?  

As a gold standard for oncology research, hundreds of millions of FFPE samples are collected and banked worldwide. These samples provide a rich source of data for identification of biomarkers in the search for early detection assays for cancer as well as diagnostics that could help direct treatment decisions and monitor treatment.  

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Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.

Posted on by Leah Cronan

PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?

Continue reading “Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.”

Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

Posted on by Leah Cronan

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

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Reliable DNA Purification from 3D Cell Cultures

Posted on by Julia Nepper

Traditionally, scientists have relied on flat, two-dimensional cell cultures grown on substrates such as tissue culture polystyrene (TCPS) to study cellular physiology. These models are simple and cost-effective to culture and process. Within the last decade, however, three-dimensional (3D) cell cultures have become increasingly popular because they are more physiologically relevant and better represent in vivo conditions.

A spheroid of ~1,000 human liver cells. Image provided by Insphero.
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T-Vector Cloning: Questions, Answers and Tips

Posted on by Leah Cronan
Blue/White colony screening helps you pick only the colonies that have your insert.

Q: Can PCR products generated with GoTaq DNA Polymerase be used to for T- vector cloning?

A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq® DNA Polymerase lacks 3’ →5’ exonuclease activity and displays terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerases (including the GoTaq® Flexi and GoTaq® G2 polymerases) will contain A-overhangs which makes them suitable for T-vector cloning with the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.

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The Future of Synthetic Biology: A Recap of iGEM 2019

Posted on by Darcia Schweitzer

After attending the iGEM Giant Jamboree last year and being completely blown away by the projects presented (check out this article or this one), I didn’t think I’d be as astonished this year. I attributed part of the awe I felt over the caliber and quality of the projects to my wide-eyed naiveté, having never attended the event before. The second time around, the “first-time” novelty long worn off, I didn’t expect to feel that same level of amazement.

I couldn’t have been more wrong.

After three days of impressive presentations, I once again felt that same astonishment as I prepared to watch the presentations of the 6 finalists. With good reason—the projects presented by the six finalists completely blew my mind!

Continue reading “The Future of Synthetic Biology: A Recap of iGEM 2019”