Nucleic Acid Analysis

Studying Autophagy in Flies Using CRISPR

Posted on by Leah Cronan

Transcribed RNA can be used to study RNA structure and how it relates to function or how proteins and RNA interact. It can also be used for gene silencing using RNAi (studied more often as a possible therapeutic option) or simply serve as a molecular standard in Real-time RT-PCR. Transcribed RNA is also used in Class 2 Clustered Regularly Interspaced Short Palindromic Repeat systems, or CRISPR.

The CRISPR system, which is naturally occurring in bacteria, has been manipulated to perform gene editing in a laboratory environment. To perform CRISPR in the laboratory environment, you need two main reagents:

  1. The Brains: Guide RNA (gRNA or sgRNA) – Small piece of RNA containing a nucleotide sequence that is capable of binding the chosen Cas Protein, and contains a portion of the sequence that can bind the DNA the researcher intends to modify – the target DNA.
  2. The Brawn: CRISPR-associated endonuclease (Cas Protein) – The protein that cleaves the target DNA; the most popular Cas protein is called Cas9. The Cas protein is guided by the (gRNA).

Guo et al. used Promega’s RiboMAX™ Large-Scale RNA Production System to produce gRNA to be used in CRISPR for their study to determine the effects of the loss of, or mutations in, a specific gene in fruit flies (1).  Atg101 is a gene that plays an important role in autophagy, an intracellular pathway for removing toxins or damaged parts of cells.

Continue reading “Studying Autophagy in Flies Using CRISPR”

Promega Scientific Applications: Expertise to Optimize Your Workflow

Posted on by Melanie Preston
Molecular biology protocols are being applied by the cannabis industry to ensure safety and improve production.

How many times have you encountered a technical problem in your work that you needed to solve? Maybe it was an issue of workflow efficiency—too many samples, but too little time for hands-on work. Or maybe there wasn’t a technology available for what you needed to accomplish, and you didn’t have time to develop something yourself. Or still, maybe you were starting into a new research area and didn’t yet have the expertise to solve the problem. Wouldn’t it be nice if you had some support to figure out a solution for these challenges? We have scientists at your service! You may already know about our top-notch team of Technical Services Scientists. They can assist you via phone, email, or chat to walk you through any technical issue, regardless of whether or not you’re using Promega products (not too many companies can say that!).

We go beyond that level of assistance with the expertise of the Scientific Applications team.

Continue reading “Promega Scientific Applications: Expertise to Optimize Your Workflow”

How Do You Solve a Problem Like Malaria?

Posted on by Mariel Mohns
malaria_researcher
Photo courtesy of NIH/NIAID

Malaria affects nearly half of the world’s population, with almost 80% of cases in sub-Saharan Africa and India. While there have been many strides in education and prevention campaigns over the last 30 years, there were over 200 million cases documented in 2017 with over 400,000 deaths, and the majority were young children. Despite being preventable and treatable, malaria continues to thrive in areas that are high risk for transmission. Recently, clinicians started rolling out use of the first approved vaccine, though clinical trials showed it is only about 30% effective. Meanwhile, researchers must continue to focus on innovative efforts to improve diagnostics, treatment and prevention to reduce the burden in these areas.

Continue reading “How Do You Solve a Problem Like Malaria?”

Cloning Modified Blunt-ended DNA Fragments into T-Vectors

Posted on by Kelly Grooms

Tailing blunt-ended DNA fragments with TaqDNA Polymerase allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors. This method also eliminates some of the requirements of conventional blunt-end cloning — Fewer steps, who can argue with that?

Blue/White colony screening helps you pick only the colonies that have your insert.
Blue/White colony screening helps you pick only the colonies that have your insert.

Continue reading “Cloning Modified Blunt-ended DNA Fragments into T-Vectors”

Meet Měnglà Virus: the newest cousin in the Ebola and Marburg virus family tree

Posted on by Mariel Mohns

Ebola virus (EBOV) and Marburg virus (MARV) are two closely-related viruses in the family Filoviridae. Filoviruses are often pathogenic, causing hemorrhagic fever disease in human hosts. The Ebola outbreak of 2014 caught the world by surprise by spreading so quickly and severely that public health organizations were unprepared. The devastating outcome was a total of over 11,000 deaths by the time the outbreak ended in 2016. Research that provides further understanding of filoviruses and their potential for transmission is important in preventing future outbreaks from occurring. But what if the outbreak comes from a virus we’ve never seen before?

fruit_bat
Měnglà virus was discovered among filoviruses isolated from Old World fruit bats (Rousettus)

All in the viral family

A recent study published in the journal Nature Microbiology provides evidence of a newly identified filovirus species. Using serum samples taken from bats, a well-known host for filoviruses, Yang et al. isolated and identified viral RNA for an unclassified viral genome sequence using next generation sequencing analysis. This new virus genome sequence was organized with the same open reading frames as other filoviruses, encoding for nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L). This new genome sequence shared up to 54% of the nucleotide sequences for the filovirus species Lloviu virus (LLOV), EBOV and MARV, with MARV being the most similar. Their analysis suggested that this novel virus should be classified within the Filoviridae family tree as a separate genus, Dianlovirus, and was named Měnglà virus (MLAV).

Continue reading “Meet Měnglà Virus: the newest cousin in the Ebola and Marburg virus family tree”

Deep in the Jungle Something Is Happening: DNA Sequencing

Posted on by Promega

This blog was written by guest blogger and 2018 Promega Social Media Intern Logan Godfrey.

Only 30 years ago, the polymerase chain reaction (PCR) was used for the first time, allowing the exponential amplification of a specific DNA segment. A small amount of DNA could now be replicated until there was enough of it to study accurately, even allowing sequencing of the amplified DNA. This was a massive breakthrough that produced immediate effects in the fields of forensics and life science research. Since these technologies were first introduced however, the molecular biology research laboratory has been the sole domain of PCR and DNA sequencing.

While an amazing revolution, application of a technology such as DNA sequencing is limited by the size and cost of DNA sequencers, which in turn restricts accessibility. However, recent breakthroughs are allowing DNA sequencing to take place in jungles, the arctic, and even space—giving science the opportunity to reach further, faster than ever before. 

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra.

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra. Photo credit: Field Projects International.

The newfound accessibility of DNA sequencing means a marriage between fields of science that were previously largely unacquainted. The disciplines of genomics and wildlife biology/ecology have largely progressed independently. Wildlife biology is practiced in the field through observations and macro-level assessments, and genomics, largely, has developed in a lab setting. Leading the charge in the convergence of wildlife biology and genomics is Field Projects International. 

Continue reading “Deep in the Jungle Something Is Happening: DNA Sequencing”

Harnessing the Power of Massively Parallel Sequencing in Forensic Analysis

Posted on by Ken Doyle

The rapid advancement of next-generation sequencing technology, also known as massively parallel sequencing (MPS), has revolutionized many areas of applied research. One such area, the analysis of mitochondrial DNA (mtDNA) in forensic applications, has traditionally used another method—Sanger sequencing followed by capillary electrophoresis (CE).

Although MPS can provide a wealth of information, its initial adoption in forensic workflows continues to be slow. However, the barriers to adoption of the technology have been lowered in recent years, as exemplified by the number of abstracts discussing the use of MPS presented at the 29th International Symposium for Human Identification (ISHI 29), held in September 2018. Compared to Sanger sequencing, MPS can provide more data on minute variations in the human genome, particularly for the analysis of mtDNA and single-nucleotide polymorphisms (SNPs). It is especially powerful for analyzing mixture samples or those where the DNA is highly degraded, such as in human remains. 

Continue reading “Harnessing the Power of Massively Parallel Sequencing in Forensic Analysis”

Overcoming Challenges When Scaling Antibody Production

Posted on by Darcia Schweitzer

Tradeoffs are a constant source of challenge in any research lab. To get faster results, you will probably need to use more resources (people, money, supplies). The powerful lasers used to do live cell imaging may well kill those cells in the process. Purifying DNA often leaves you to choose between purity and yield.

Robot performing autosampling

Working with biologics also involves a delicate balancing act. Producing compounds in biological models rather than by chemical synthesis offers many advantages, but it is not without certain challenges. One of those tradeoffs results from scaling up; the more plasmid that is produced, the greater probability of endotoxin contamination.

Continue reading “Overcoming Challenges When Scaling Antibody Production”

Is MPS right for your forensics lab?

Posted on by Promega

Today’s post was written by guest blogger Anupama Gopalakrishnan, Global Product Manager for the Genetic Identity group at Promega. 

Next-generation sequencing (NGS), or massively parallel sequencing (MPS), is a powerful tool for genomic research. This high-throughput technology is fast and accessible—you can acquire a robust data set from a single run. While NGS systems are widely used in evolutionary biology and genetics, there is a window of opportunity for adoption of this technology in the forensic sciences.

Currently, the gold standard is capillary electrophoresis (CE)-based technologies to analyze short tandem repeats (STR). These systems continue to evolve with increasing sensitivity, robustness and inhibitor tolerance by the introduction of probabilistic genotyping in data analysis—all with a combined goal of extracting maximum identity information from low quantity challenging samples. However, obtaining profiles from these samples and the interpretation of mixture samples continue to pose challenges.

MPS systems enable simultaneous analysis of forensically relevant genetic markers to improve efficiency, capacity and resolution—with the ability to generate results on nearly 10-fold more genetic loci than the current technology. What samples would truly benefit from MPS? Mixture samples, undoubtedly. The benefit of MPS is also exemplified in cases where the samples are highly degraded or the only samples available are teeth, bones and hairs without a follicle. By adding a sequencing component to the allele length component of CE technology, MPS resolves the current greatest challenges in forensic DNA analysis—namely identifying allele sharing between contributors and PCR artifacts, such as stutter. Additionally, single nucleotide polymorphisms in flanking sequence of the repeat sequence can identify additional alleles contributing to discrimination power. For example, sequencing of Y chromosome loci can help distinguish between mixed male samples from the same paternal lineage and therefore, provide valuable information in decoding mixtures that contain more than one male contributor. Also, since MPS technology is not limited by real-estate, all primers in a MPS system can target small loci maximizing the probability of obtaining a usable profile from degraded DNA typical of challenging samples.

Continue reading “Is MPS right for your forensics lab?”

Of Mice and Microbes: The Science Behind Food Analysis

Posted on by Julia Nepper

In general, people like to know that their food is what the label says it is. It’s a real bummer to find out that beef lasagna you just ate was actually horsemeat. Plus, there are many religious, ethical and medical reasons to be cognizant of what you eat. Someone who’s gluten intolerant and Halal probably doesn’t want a bite of that BLT.

Labels don’t always accurately reflect what is in food. So how do we confirm that we are in fact buying crab, and not whitefish with a side of Vibrio contamination?

For the most part, it comes down to separation science. Scientists and technicians use various chromatographic methods, such as gas chromatography, liquid chromatography, and mass spectrometry, to separate the complex mixture of molecules in food into individual components. By first mapping out the molecular profile of reference samples, they can then take an unknown sample and compare its profile to what it should look like. If the two don’t match up, an analyst would assume that the unknown is not what it claims to be. Continue reading “Of Mice and Microbes: The Science Behind Food Analysis”