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.
The elk tooth is small and ancient, with a crude hole bored through the top. It was likely worn as a pendant, but worn by whom? Was the owner male or female? Where did they come from? Did the pendant indicate their social status, mark a significant accomplishment, was it a gift, or was it worn as an expression of individuality?
Artifacts such as personal ornaments and tools play a pivotal role in helping us understand the migration, behavior and cultures of ancient peoples. To date, this information has stopped short of providing insight into things like the biological sex or genetic ancestry of the individuals who may have worn or used these items, and thus limited our ability to accurately characterize societal roles and behaviors. Recent advances in DNA techniques and technologies, and one little pendant, might be changing that.
There’s a certain group of people (including this blog post author) who derive no small amount of amusement from seeing stock photos of DNA and pointing out flaws in the structure. It’s even more amusing when these photos are used in marketing by life science companies. The most common flaw: the DNA molecule is a left-handed double helix.
What does that even mean? DNA, like many organic chemicals in biology, is a chiral molecule. That is, it can exist in two structural forms that are mirror images of each other but are not superimposable (enantiomers). Just like your left and right hands are mirror images of each other, the two DNA structures are left-handed and right-handed double helices. The DNA double helix is chiral, because its building blocks (nucleotides) are chiral.
It can be challenging, at first glance, to tell whether an image of DNA is left-handed or right-handed. Various helpful hints are available; however, the one that I’ve found easiest to remember is described in a blog post by Professor Emeritus Larry Moran at the University of Toronto:
Imagine that the double helix is a spiral staircase, and you’re walking down the staircase. If you’re turning to the right as you descend, the DNA structure is right-handed; if turning to the left, it’s left-handed. In the image shown earlier, the DNA molecule on the right is a right-handed double helix, while its mirror image is left-handed.
Several different types of nucleic acids can be found circulating in human biofluids. Fragmented DNA and RNA are now routinely purified from plasma and other bodily fluids. These types of nucleic acids need to be purified from a cell-free fraction of the biofluids to ensure that the isolated nucleic acids are truly circulating and not from intact cells. In this blog post, we will learn a bit more about circulating nucleic acids (CNA) and how they can be used as biomarkers in research.
In oncology, tissue biopsies are commonly fixed in formalin and embedded in paraffin (FFPE). These FFPE samples can be used with immunohistochemical or molecular analysis for identifying biomarkers that guide the diagnosis and therapeutic management of patients. This fixation technique allows long-term storage of samples but impacts the integrity of nucleic acids. This makes extracting DNA and RNA from FFPE tissues in sufficient quantity and quality for molecular analysis techniques such as NGS analyses challenging for molecular oncology laboratories.
“At Rennes University Hospital, we receive many lung cancer samples with little material available, or samples of poor quality. The nucleic acid extraction step is therefore critical to get good yield. We have seen that it had a direct impact on the success of downstream analysis,” said Dr. Alexandra Lespagnol. Lespagnol is the Technical Manager of the Molecular Genetics of Cancer core lab at the University Hospital of Rennes in France.
In order to accommodate the increasing number of samples that needed to be analyzed, the Molecular Genetics of Cancer core lab of the University Hospital of Rennes initiated an automation project for extracting DNA from FFPE tissues. The lab also wanted to improve sample tracking and reproducibility of their results.
Tracking the spread of COVID-19 has been a tremendous challenge throughout the pandemic, but doing so is a key step toward containing the virus. Many communities have relied on patient testing and contact tracing, with limited success. In search of better methods, some countries have made inroads in a different form of disease surveillance: wastewater-based epidemiology (WBE). This approach involves testing wastewater for the presence of pathogens, primarily through DNA and RNA analysis, and has proved to be an accurate and highly effective way to keep tabs on the prevalence and progression of COVID-19 at the population level.
Switzerland is among those countries that have implemented WBE in their efforts to stay ahead of the pandemic. Since WBE first emerged in 2020 as a promising tool, several Swiss laboratories undertook wastewater testing, and protocols were established early.
“At the beginning, the methods to actually detect coronavirus in wastewater were rather laborious and complicated, and involved a lot of resources,” said Dr. Claudia Bagutti, microbiologist and molecular biologist in the State Laboratory of Basel-City, Switzerland.
Bagutti heads a small team performing applied biosafety research. In 2020, her lab was tasked with developing an assay for detecting COVID-19 in wastewater. However, the available methods were prohibitively complex and resource intensive.
In the meantime, researchers at Promega recognized that Promega products and methodologies could potentially be applied to WBE and set to work developing simpler and more efficient method for wastewater analysis. In the spring of 2021, Bagutti’s team decided to try adopting this method.
“Promega had a very nice method which was less laborious and much easier to handle, and that’s why we gave it a try,” said Bagutti.
In the ensuing study, Bagutti and her team analyzed effluent from the catchment area of one municipal wastewater plant in Switzerland. They examined the total wastewater output of around 270,000 people. Viral RNA was extracted using Promega’s Maxwell® RSC Environ Wastewater TNA Kit. The number of RNA copies present, representing the overall concentration of COVID-19 in each sample, was determined via quantitative reverse transcriptase (RT-qPCR) using the GoTaq® Enviro Wastewater SARS-CoV-2 Systems, also from Promega. The viral RNA was subsequently sequenced with next generation sequencing, and the results correlated quite well with the COVID-19 cases in the catchment area. Remarkably, this study detected the Omicron variant in a wastewater sample one day prior to the first reported case identified through patient testing.
“We observed a similar spread to most other western countries with respect to the time of the first discovery of these variants,” said Bagutti. “We were also able to demonstrate the presence [of Omicron] in the wastewater before it came up in a sample of a COVID-19 patient test, which of course shows the usefulness of wastewater monitoring for the prediction of new variants and infection dynamics.”
WBE is especially promising in that it provides population-level data independent of patient testing. Health departments can be alerted to the presence of COVID-19 earlier than would otherwise be possible with traditional testing and can take precautions to contain the spread. In creating a more user-friendly method for wastewater analysis, Promega has opened the door for more laboratories to conduct WBE, which could provide communities around the world with the information they need to preempt the progression of COVID-19.
“The Promega method is very straightforward to handle,” said Bagutti. “It only takes a small volume of wastewater, which makes it handy. It’s less time-consuming compared to the methods which were in the literature at the beginning of the pandemic, and it just works very well. We also did experience great support from Promega.”
At this point, much of the wastewater analysis performed in Switzerland is done with the Promega method, including in federal, state or private labs. The swift advance of WBE in Switzerland speaks to the colossal effort put forth both by Promega researchers in developing the necessary products and methodologies, as well by those labs that have made use of Promega’s products to monitor COVID-19 in wastewater.
“It’s really been a success story for us, from the beginning,” said Bagutti.
Learn more about Promega’s work with wastewater-based epidemiology.
Nucleic acid extraction is a time-consuming, resource-intensive process, but it doesn’t have to be. Automated systems are becoming more and more accessible and often can be operated with simple “plug and play” kits, freeing valuable resources
With these systems increasingly within reach, perhaps you’re thinking about introducing automated nucleic acid extraction into your lab. As you consider your options, here’s eight reasons why we think you should automate your nucleic extraction workflows.
1. Reach your project milestones and publish faster.
In the fast-paced, competitive environment of research and technology development, efficiency is key to reaching project milestones and publishing your work. Managing your resources effectively–especially time–can help you reach those goals.
Time spent on manual nucleic acid extractions is time lost on parallel work, which cuts down productivity. Automation is not only often faster than manual preparations, but it also frees your team to do more valuable hands-on work.
As an example, the Maxwell® RSC cuts 40 minutes of hands-on-time per 16 samples. As the number of samples scales to 96 and beyond, liquid handlers like the Hamilton Star or Tecan Fluent can save many hours of hands-on-time per day.
A new study, published in the Journal of Molecular Diagnostics (1), highlights the potential of using long mononucleotide repeat (LMR) markers for characterizing microsatellite instability (MSI) in several tumor types. The paper is a result of a collaborative effort between researchers from Johns Hopkins University and Promega to evaluate the performance of a panel of novel LMR markers for determining MSI status of colorectal, endometrial and prostate tumor samples.
Microsatellite instability (MSI) is the accumulation of insertion or deletion errors at microsatellites, which are short tandem repeats of DNA sequences found throughout the genome. MSI in cancerous cells is the result of a functional deficiency within one or more major DNA mismatch repair proteins (dMMR). PCR-based MSI testing is a commonly used method that can help understand a tumor’s genomic profile as it relates to MMR protein function.
Historically, MSI has been a biomarker associated with Lynch syndrome, the hereditary predisposition to colorectal and certain other cancers. In recent years, research interest in MSI has exploded, driven by the discovery that its presence in tumor tissue can be predictive of a positive response to anti-PD-1 immunotherapies (2,3).
Imagine that you’re putting together a large, complex jigsaw puzzle, comprising thousands of exceptionally small pieces. You lay them all out and attempt to make sense of them. It would be far easier to assemble this puzzle were the pieces larger, containing more of the image advertised on the box. The same can be said when sequencing a genome.
Traditional short-read or next-generation sequencing relies on DNA spliced into small fragments (≤300 base pairs) and then amplified. While useful for detecting small genetic variants like single-base changes to the DNA, this type of sequencing can fail to illuminate larger variations (typically over 50 base pairs) in the genome. Long-read sequencing, or third generation sequencing, allows more accurate genome assemblies, facilitating better detection of structural variants like copy number variations, duplications, translocations and inversions that are too large to identify with short-read sequencing. Long-read sequencing has the capability to fill in “dark regions” of a genome that are unfinished and can be used to assemble larger, more complex genomes using longer fragments of DNA, or high-molecular weight (HMW) DNA.
This blog was written by guest writers Paraj Mandrekar (Technical Services Scientist 3) and Michelle Mandrekar, (Research Scientist 4).
Here are some designer’s notes comparing the Maxwell® RSC Blood DNA and the Maxwell® RSC simplyRNA kit chemistries for nucleic acid extraction.
The Maxwell RSC Blood DNA Kit and Maxwell RSC simplyRNA Blood Kit were both developed from the same non-silica-based purification chemistry and use the same underlying paramagnetic particle. This chemistry is characterized by an extreme binding capacity (the capacity of nucleic acid that can be bound on the particle), leading to both chemistries being capable of isolating large amounts of nucleic acid volumes and then eluting into relatively small volumes (50 µL). It is not unusual with either chemistry to have isolates that exceed 100 ng/µL. Although the chemistries have several similarities, there are some important distinctions between how the two chemistries were designed that influence which kit you choose for your nucleic acid extraction.
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