“The Human Placenta,” or “Why I Love Science Writing”

Posted on by Jordan Villanueva

Have you read last week’s breaking story about the microbiome of the human placenta? Wait, stop, don’t run away to Google it! I’ll tell you all about it – this is a science blog, remember?

I’m asking because as I started reading about the topic in preparation for writing this blog post, I noticed two things. First, as a science writer who tries to stay well-connected with what’s going on in the world of biology research, it would have been nearly impossible for me to avoid this story. I get eight or nine daily digest emails from scientific publications every day, and I think over the course of last week, every single one came with a headline related to the placenta study. (Of course, I read them all. And the Nature study they were based on.)

Second, I noticed that each story I read had a slightly different angle on covering the research. As scientists, we like to believe that science is, well, just science. It’s factual. We pore over the data and reach a conclusion. If we aren’t sure of something, we search the journals. The story, if there is one, is about methods and controls, protocols and reagent quality. However, when information about that research is communicated broadly, outside of the journals, we can get a different impression based on how the author frames their article. Continue reading ““The Human Placenta,” or “Why I Love Science Writing””

qPCR: The Very Basics

Posted on by Michele Arduengo
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.
qPCR monitors amplification in real and allows you to measure starting material.

For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.

First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.

End-Point PCRqPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point)Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNAMeasures how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments requiredMore expensive; requires special instrumentation
Basic molecular biology techniqueRequires slightly more technical prowess

Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real-time and detect the number of copies in the starting material can quantitate gene expression, measure DNA damage, and quantitate viral load in a sample and other applications.

Anytime that you are performing a reaction where something is copied over and over in an exponential fashion, contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.

  1. Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves and change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. Use reaction “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions. Because you are pipetting larger volumes to make the reaction master mix, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
  5. Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.

These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.

If you are looking for more information regarding qPCR, watch this supplementary video below.

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

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Optimizing PCR: One Scientist’s Not So Fond Memories

Posted on by Kelly Grooms

primer_tubesThe first time I performed PCR was in 1992. I was finishing my Bachelors in Genetics and had an independent study project in a population genetics laboratory. My task was to try using a new technique, RAPD PCR, to distinguish clonal populations of the sea anemone, Metridium senile. These creatures can reproduce both sexually and asexually, which can make population genetics studies challenging. My professor was looking for a relatively simple method to identify individuals who were genetically identical (i.e., potential clones).

PCR was still in its infancy. No one in my lab had ever tried it before, and the department had one thermal cycler, which was located in a building across the street. We had a paper describing RAPD PCR for population work, so we ordered primers and Taq DNA polymerase and set about grinding up bits of frozen sea anemone to isolate the DNA. [The grinding process had to be done using a mortar and pestle seated in a bath of liquid nitrogen because the tissue had to remain frozen. If it thawed it became a disgusting mass of goo that was useless—but that is a topic for a different blog.] Since I had never done any of the procedures before, my professor and I assembled the first set of reactions together. When we ran our results on a gel, we had all sorts of bands—just what he was hoping to see. Unfortunately, we realized that we had added 10X more Taq DNA polymerase than we should have used. I repeated the amplification with the correct amount of Taq polymerase, and I saw nothing. Continue reading “Optimizing PCR: One Scientist’s Not So Fond Memories”

When Five Hundred Tigers Are Not Enough—Ten Years Later

Posted on by Promega

Ten years ago, I wrote about the distressing news of lack of genetic diversity in the wild Amur tiger population. International Tiger Day seemed like a good time to check in on what progress has been made to both sustain and establish wild tiger populations worldwide. In 2010, 13 tiger range countries (TRC) committed to a goal of doubling the world’s tiger population by 2022.

Amur Tiger.

That timeline was an ambitious goal, as highlighted by a report published in PLOS One in November of 2018 (1). The authors assessed the recovery potential of 18 sites identified under the World Wide Fund for Nature’s (WWF) Tigers alive initiative. The recovery system has several parts: A source site with higher density of tigers that the area around it and has a legal framework that does (or will) protect the tiger population; a recovery site that has a lower density of tigers than the surrounding regions, has the ability to support more tigers but is not as supported as a source site; and a support region that connects a source and recovery site. These different site types all require different levels of management, available resources and legal protections, but they need to be managed in a coordinated way.

Aside from what is needed to manage these recovery sites, there are also other things that need to exist to support recovery of tiger populations. Some of these include support from local populations and governments, as well as environmental requirements such as breeding habitats and prey populations. For 15 of the 18 sites it is the prey population that is the sticking point. Recovery of prey populations is a slow process. The authors concluded that there need to be a commitment to achieving a realistic recovery of tiger populations, even if we miss the 2022 goal.

The fate of the wild tiger is still tenuous. Only time will tell if the interventions that are being implemented can be realized in time.

Reference

  1. Abishek, H. et al. (2018) Recovery planning towards doubling wild tigers Panthera tigris numbers: Detailing 18 recovery sites from across the range. PLOS One 13. e0207114. published online

Think Restriction Enzymes are so last decade? Not so fast!

Posted on by Leah Cronan
Ribbon diagram of EcoRI homodimer bound to doublestranded DNA
Ribbon diagram of EcoRI homodimer bound to doublestranded DNA

Restriction enzymes sometimes get a lot of flak. In the not-so-distant past, they were the workhorses of molecular biology. Restriction enzymes played a huge role in developing early DNA sequencing techniques. They chop DNA in a predictable manner, which makes cutting and pasting genes of interest manageable and relatively easy, enabling the development of  genetic engineering and recombination technologies. These technologies are now moving beyond restriction enzymes toward more modern methods, with the most talked-about method being CRISPR /Cas9. As technology continues to advance at such a rapid pace, restriction analysis  and other “ancient” technologies feel antiquated. But this is not necessarily the case.

Continue reading “Think Restriction Enzymes are so last decade? Not so fast!”

CRISPR/Cas9, NanoBRET and GPCRs: A Bright Future for Drug Discovery

Posted on by Ken Doyle

GPCRs

G protein-coupled receptors (GPCRs) are a large family of receptors that traverse the cell membrane seven times. Functionally, GPCRs are extremely diverse, yet they contain highly conserved structural regions. GPCRs respond to a variety of signals, from small molecules to peptides and large proteins. Many GPCRs are involved in disease pathways and, not surprisingly, they present attractive targets for both small-molecule and biologic drugs.

In response to a signal, GPCRs undergo a conformational change, triggering an interaction with a G protein—a specialized protein that binds GDP in its inactive state or GTP when activated. Typically, the GPCR exchanges the G protein-bound GDP molecule for a GTP molecule, causing the activated G protein to dissociate into two subunits that remain anchored to the cell membrane. These subunits relay the signal to various other proteins that interact with or produce second-messenger molecules. Activation of a single G protein can result, ultimately, in the generation of thousands of second messengers.

Given the complexity of GPCR signaling pathways and their importance to human health, a considerable amount of research has been devoted to GPCR interactions, both with specific ligands and G proteins.

Continue reading “CRISPR/Cas9, NanoBRET and GPCRs: A Bright Future for Drug Discovery”

Empowering Communities with the Light of the Sun

Posted on by Promega

Today’s blog brought to you by Julia Nepper, a Promega science writer guest blogging for the BioPharmaceutical Technology Center Institute (BTC Institute)!

“We all benefit from STEM role models. When students from underrepresented populations meet and learn about STEM professionals of color, they can see themselves as the scientists and engineers of the future. Fun, engaging science programming for children is also essential to light the spark for the next generation. A Celebration of Life, the partnership between the BTC Institute and the African American Ethnic Academy, two community nonprofits, has combined these 2 objectives for over twenty years.” according to Barbara Bielec, K-12 Program Director.

This year, the theme of the program is Sunsational!, with a number of activities related to the sun, solar energy, and STEM careers. As part of the program, students heard talks from several STEM professionals of color about their work. Mehrdad Arjmand, co-founder of solar energy company NovoMoto, was one of those speakers.

Dr. Arjmand was born and raised in Iran. His path to becoming a mechanical engineer began as a child, with him “destroying a lot of equipment” in his house. After completing his undergraduate education, he came to the States to pursue a PhD at the University of Wisconsin-Madison, where he met Aaron Olson, a student who was born in the Democratic Republic of Congo. These two discovered a shared passion for starting a business and helping their communities, which led directly to the founding of NovoMoto. The name derives from Portuguese for “new” (novo) and Lingala—a language spoken in Congo—for “fire” (moto). Continue reading “Empowering Communities with the Light of the Sun”

Restriction Enzyme Digestion: Capabilities and Resources

Posted on by Promega
Image of someone pipetting while setting up a restriction enzyme digestion

Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences.  These enzymes are the workhorse in many molecular biology applications such as cloning, RFLP, methylation-specific restriction enzyme analysis of DNA, etc.  Restriction enzymes with enhanced capabilities can help you streamline and shorten these workflows and improve success of restriction enzyme digestion.

A subset of Promega restriction enzymes offer capabilities that  include rapid digestion of DNA in 15 minutes or less, ability to completely digest DNA directly in the GoTaq® Green Master Mix, and Blue/White Cloning Qualification which allows for rapid, reliable detection of transformants.

To learn more about restriction enzymes and applications, check out Restriction Enzyme Resource on the web. The resource provides everything from information on restriction enzyme biology to practical information on how to set up and design a restriction enzyme digestion. This resource also contains useful online tools, including the Restriction Enzyme Tool, to help you use enzymes more effectively. It helps you choose the best reaction buffer for double digests, find the commercially available enzyme that cuts your sequence of interest, find compatible ends, and search for specific information on cut site, overhang isoschizomers and neoschizomers by enzyme name.

For added convenience, you can download the mobile app available for iOS devices and use the Restriction Enzyme Tool to plan your next digest.

For additional information regarding Restriction Enzyme Digest, reference the supplementary video below.

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A Quick Method for A Tailing PCR Products

Posted on by Isobel Maciver
PCR experiment and products, pipette tip, tube in researcher's hand.
PCR is a common technique used in research labs to amplify DNA.

Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.

Continue reading “A Quick Method for A Tailing PCR Products”

Meet the Mighty Masked Masters of Measurement

Posted on by Michele Arduengo

Scientific investigation is an iterative process, for which reproducibility is key. Reproducibility, in turn, requires accuracy and precision—particularly in measurement. The unsung superheroes of accuracy and precision in the research lab are the members of your local Metrology Department. According to Promega Senior Metrologist, Keela Sniadach, it’s good when the metrology department remains unsung and behind the scenes because that means everything is working properly.

Holy Pipettes, Scientists! We have a metrology department?! Wait…what’s metrology again?

Callibration technician checks out a multipipettor

Metrology (the scientific study of measurement) got its start in France, when it was proposed that an international length standard be based on a natural source. It was from this start that the International System of Units (SI), the modern metric system of measurement, was born.

Metrology even has its own day: May 20, which is the anniversary of the day that the International Bureau of Weights and Measures (BIPM) was created by the Meter Convention in Paris in 1875. The job of BIPM is to ensure worldwide standards of measurement.

For life scientists, metrology centers around making sure the equipment used everyday—from pipettes to heating blocks to centrifuges—is calibrated and measuring correctly.

Continue reading “Meet the Mighty Masked Masters of Measurement”