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.
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.
Foodborne disease affects almost 1 in 10 people around the world annually, and continuously presents a serious public health issue (9).
More than 200 diseases have evolved from consuming food contaminated by bacteria, viruses, parasites, and chemical substances, resulting in extensive increases in global disease and mortality rates (9). With this, foodborne pathogens cause a major strain on health-care systems; as these diseases induce a variety of different illnesses characterized by a multitude of symptoms including gastrointestinal, neurological, gynecological, and immunological (9,2).
But why is food contamination increasing?
New challenges, in addition to established food contamination hazards, only serve to compound and increase food contamination risks. Food is vulnerable to contamination at any point between farm and table—during production, processing, delivery, or preparation. Here are a few possible causes of contamination at each point in the chain (2):
Production: Infected animal biproducts, acquired toxins from predation and consumption of other sick animals, or pollutants of water, soil, and/or air.
Processing: Contaminated water for cleaning or ice. Germs on animals or on the production line.
Delivery: Bacterial growth due to uncontrolled temperatures or unclean mode of transport.
Preparation: Raw food contamination, cross-contamination, unclean work environments, or sick people near food.
Further emerging challenges include, more complex food movement, a consequence of changes in production and supply of imported food and international trade. This generates more contamination opportunities and transports infected products to other countries and consumers. Conjointly, changes in consumer preferences, and emerging bacteria, toxins, and antimicrobial resistance evolve, and are constantly changing the game for food contamination (1,9).
Hence, versatile tests that can identify foodborne illnesses in a rapid, versatile, and reliable way, are top priority.
In the fifty years since the first reported transformation of recombinant plasmids into bacteria (1), plasmid cloning has become one of the pillars of synthetic biology research and manufacturing biopharmaceuticals.
But purifying plasmids is no small feat. It can often take hours of hands-on time to go from culture to eluate with low-throughput and time-sensitive manual methods. Automating plasmid purification is the way to go, whether you’re isolating a single plasmid from a large volume culture or creating a library of thousands of different constructs.
We recently posted a blog about Proteinase K, a serine protease that exhibits broad cleavage activity produced by the fungus Tritirachium album Limber. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. In that previous blog we focused on its use to remove RNase and DNase activities. However, the stability of Proteinase K in urea and SDS and its ability to digest native proteins make it useful for a variety of applications. Here we provide a brief list of peer-reviewed citations that demonstrate the use of proteinase K in DNA and RNA purification, protein digestion in FFPE tissue samples, chromatin precipitation assays, and proteinase K protection assays: Continue reading “Proteinase K: An Enzyme for Everyone”
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”
Aberrant methylation events have significant impacts in terms of incidence of cancer and development disregulation. Researchers studying DNA methylation are often working with DNA from “difficult” tissues such as formalin-fixed, paraffin embedded tissues, which characteristically yield DNA that is more fragmented than that purified from fresh tissue. Traditional methods for bisulfite conversion involve a long protocol, harsh chemicals, and generally yield highly fragmented DNA. The DNA fragmentation may significantly impact the utility of the converted DNA in downstream applications such as bisulfite-specific PCR or bisulfite sequencing.
An ideal bisulfite conversion system enables complete conversion of a DNA sample in a short period of time, provides high yield of DNA, minimally fragments the DNA, works on a wide range of input DNA amounts (from a wide variety of sample types), and, while we’re at it, is easy to use and to store. Whew! That’s quite the list.
Yesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.
This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.
The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading “DNA Purification, Quantitation and Analysis Explained”
Last week I read an article in Wired Science that described how an outbreak of antibiotic resistant Klebsiella pneuomiae was tracked in real-time at an NIH hospital using DNA sequencing technologies. The article described how whole genome sequencing of disease isolates and environmental samples from the hospital was used to track the source and spread of the outbreak.
The scientists monitoring the outbreak tracked spontaneous random mutations in the K. pneumoniea DNA sequence to determine that the outbreak was caused by a single source, and to track the spread of the organism within the hospital. The sequencing information helped investigators identify when and where infection occurred, and also to track transmission of the infection from person-to-person. It also revealed that the order of transmission was different from the order in which the cases presented with symptoms, and helped identify how the organism was spread between individuals.
The article describes how epidemiology, infection control and sequence identification were used together to influence outcome in this situation, but also shows the power of whole genome sequencing to find and track subtle differences between isolates that could not have been identified in any other way.
To me, this is a powerful illustration of just how far DNA sequencing has come over the last few years. Not so long ago, the idea of sequencing the entire genome of numerous disease isolates during an outbreak would have been almost laughable—an idea confined to episodes of the X-files or to science fiction stories. Now, thanks to advanced automated sequencing technologies and the computing power to analyze the results, it is doable within a reasonable timeframe for hospitals with access to the right facilities. Although this type of investigation is still beyond the capabilities of most hospitals, the costs and turnaround times for sequencing are coming down rapidly as new technologies capable of faster, cheaper analysis become available.
We have come a very long way since the days when DNA sequencing was a laborious process involving pouring a gel, running samples,and manually reading the resulting autoradiogram hoping to get a read of 50–100 bases. My reading of the wired article prompted me to find out more about the newer types of sequencing technology available today. Here’s what I learned about each: Continue reading “DNA Sequencing from AutoRads and Gels to Nanopores”
This post is provided as a general introduction to common laboratory methods for determining the yield and purity of purified DNA samples. DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are convenient, but have varying requirements in terms of equipment needed, ease of use, and calculations to consider. Continue reading “Methods for Determining DNA Yield and Concentration”
In my last entry, I gave a little summary of one of many techniques that are used to study DNA methylation patterns in a loci-specific fashion using the COBRA technique. This time, we’ll take a look at a high-throughput, genome-wide method for analyzing DNA methylation status using a next generation sequencing approache called bisulfite sequencing, or Bis-Seq. Continue reading “Bisulfite Conversion and Next Gen Sequencing”
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