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”
Over a hundred years ago William B Coley, the “Father of Immunotherapy”, discovered that injection of bacteria or bacterial toxins into tumors could cause those tumors to shrink. The introduction of bacteria had the side-effect of stimulating the immune system to attack the tumor. The field of cancer immunotherapy research—which today includes many different approaches for generating anti-tumor immune responses—originated with these early experiments.
Use of bacteria is one way to stimulate the immune system to attack cancer cells, others include use of cytokines, immune checkpoint blockades and vaccines. This Nature animation provides a simple overview of these methods.
Pipettes are such a routine part of everyday life in the lab that it can be easy to take them for granted. Their accuracy is vital, and there are many things we can adopt as best practices for success. Here are a few tips (no pun intended) gathered from around the Web by Kim Steinhauser of the Promega Metrology Department–the group charged with keeping our pipettes and other lab equipment functional and accurate. Continue reading “How to Take Care of Your Pipettes”
Yesterday, a series of 27 papers representing the most comprehensive genomic analysis of human cancers to date was published in Cell Press journals.
The collection constitutes the final outputs from the Cancer Genome Atlas (TCGA) project, a collaboration between the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) involving analysis of over 11,000 tumors representing 33 different cancers. The many research teams involved analyzed tumor DNA, mRNA, miRNA and chromatin, comparing them to matched normal cellular genomes to perform a complete molecular characterization of cancer-specific changes. The results have been presented with much hope that open access to this type of comprehensive analysis will build on recent advances in understanding tumor biology and spur further progress in developing new approaches to treatment. (See this news item for more detail).
The Pan-Cancer Atlas results are collected on a cell.com portal, where they are presented in three collections grouped by topic: Cell of Origin, Oncogenic Processes and Signaling Pathways. Each collection is accompanied by a “Flagship” paper introducing the topic and summarizing the findings. It seems fitting that these findings have been published in #HumanGenomeMonth. This comprehensive analysis of the genomic and metagenomic profiles of tumors illustrates one powerful application of the type of genomic analysis pioneered by the original Human Genome Project, and shows just how much has been made possible since the initial publication of the human genome fifteen years ago. Continue reading “The Pan-Cancer Atlas: “The End of the Beginning””
Real-time, up-to-the-minute access to information provides new opportunities for scientists to monitor cellular events in ever more meaningful ways. Real-time cytotoxicity and cell viability assay reagents now allow constant monitoring of cell health status without the need to lyse or remove aliquots from plates for measurement. With a real-time approach, data can be collected from cell cultures or microtissues at multiple time points after addition of a drug compound or other event, and the response to treatment continually observed.
The CellTox™ Green assay is a real-time assay that monitors cytotoxicity using a fluorescent DNA binding dye, which binds DNA released from cells upon loss of membrane integrity. The dye cannot enter intact, live cells and so fluorescence only occurs upon cell death, correlating with cytotoxicity. Here’s a quick overview showing how the assay works:
More Data Using Fewer Samples and Reagents
The ability to continually monitor cytotoxicity in this way makes it easy to conduct more than one type of analysis on a single sample. Assays can be combined to determine not only the timing of cytotoxicity, but to also understand related events happening in the same cell population. As long as the readouts can be distinguished from one another multiple assays can be performed in the same well, providing more informative data while using less cells, plates and reagents.
Combining assays in this way can reveal critical information regarding mechanism of cell death. For example, assay combinations can be used to determine whether cells are dying from apoptosis or necrosis, or to distinguish nonproliferation from cell death. Combining CellTox Green with an endpoint luminescent caspase assay or a real-time apoptosis assay allows you to determine whether observed cytotoxic effects are due to apoptosis. Cytotoxic and anti-proliferative effects can be distinguished by combining the cytotoxicity assay with a luminescent or fluorescent cell viability assay. Continue reading “A Better Way to Understand How and Why Cells Die”
Cell viability assays are a bread-and-butter method for many researchers using cultured cells —everyday lab tools that are a part of many newsworthy papers, but rarely make news themselves.
Over time, cell viability assays have become easier to use and more “plug ‘n play”. Among modern assays, luminescent plate-reader based systems have been a favorite for several years because of their superior sensitivity, robustness, simple protocols and uncomplicated equipment requirements (all you need is a plate-reading luminometer). These qualities combine to allow easy scalability and adaptability from bench research to high throughput applications.
CellTiter-Glo® Luminescent Cell Viability Assay is an accepted go-to viability assay for many researchers. The assay measures ATP as an indicator of metabolically active cells. A quick search on Google Scholar returns 3,990 CellTiter-Glo results for 2017 and over 500 so far in January and February of 2018. A sampling of these recent publications gives a snapshot of some of the ways the CellTiter-Glo assay is used to support key areas of research today.
Does a treatment kill cells?
The obvious application of a cell viability assay is to understand whether cells are alive. In cancer research, the CellTiter-Glo assay is often used to confirm killing of tumor cells and to verify that normal cells survive. Therefore, these assays are a key part of the evaluation and screening of drug candidates and other therapies for cancer. Many papers reporting use of CellTiter-Glo are developing and evaluating the effectiveness of novel anti-cancer treatments. Continue reading “A Cell Viability Assay for Today”
Salmonella. Streptococcus. Shigella. The most well-known bacteria are those that cause disease. Our relationship with them is one of combat. With good reason, we look for ways to avoid encountering them and to eliminate them when we do meet.
But not all bacteria are bad for us. Of course we have known for years that we are colonized by harmless bacteria, but recently, studies on the human microbiome have revealed many surprising things about these bacterial tenants. Studies are showing that the teeming multitudes of organisms living in and on the human body are not just harmless bystanders, but complex, interrelated communities that can have profound effects on our health.
Three studies published last week in Science add more to the growing body of microbiome surprises, showing that certain gut bacteria are not only good for us, but may even be required for the effectiveness of some anti-cancer immunotherapies.
Although techniques for DNA analysis of forensic samples have evolved considerably in recent years, the methods used to identify particular body fluids in forensic casework have remained relatively unchanged over the same time period. This year, one of the workshops offered at the International Symposium on Human Identification (ISHI; to be held in Seattle from October 2-5), will be focused on current and emerging techniques for body fluid identification that promise change—applying molecular genetics and proteomics analysis to the problem of body fluid identification.
According to the ISHI conference website, the purpose of the workshop is to “highlight current serology methods using critical case examples while also exploring emerging methods that could complement or replace these traditional techniques”. Continue reading “ISHI 28 Workshop: Towards Better Solutions for Body Fluid Identification”
Recently a FaceBook friend of mine (who is not a scientist) shared a video from WIRED Science where the concept of CRISPR is explained at 5 Levels of Difficulty— for a 7 year old, a teenager, a college student, a grad student and a CRISPR expert.
First it was pretty amazing to me that my non-scientist friends are interested enough in learning about CRISPR to share this type of information—perhaps showing just how popular and exciting the method has become. People outside the scientific field are hearing a lot about it, and are curious to know more.
This video does a great job of explaining the technique for all its intended audiences. It also is a nice illustration of how to share information in an easily understandable format. Even with the 7 year old and 14 year old, the information is shared in a conversational way, with everyone involved contributing to sharing information about CRISPR.
Really nice. Here’s the WIRED video:
Back in 2015 the Ice Bucket Challenge brought Amyotrophic Lateral Sclerosis (ALS) to public attention, initiating worldwide pleas for more funding of research toward a cure for this fatal disease, which is characterized by progressive degeneration of motor neurons. In spite of many efforts over the last few decades, the precise cause of ALS is still unknown.
The complexity of the problem of ALS pathogenesis is highlighted in the review “Decoding ALS: from genes to mechanism” published in Nature in November 2016. The review highlights a long list of genetic factors implicated in ALS, grouping them into genes affecting protein quality control, RNA stability/function, and the cytoskeletal structure of neuronal cells.
Mutations in the antioxidant enzyme superoxide dismutase (SOD1) were the first to be associated with ALS. According to the review, more than 170 SOD1 mutations causing ALS have since been identified. Many of these mutations are thought to result in misfolding of SOD1, contributing to toxicity when the misfolded protein accumulates within the cell.
A paper by Oh-hashi et al., published in Cell Biochemistry and Function in October 2016 used the NanoBiT protein complementation assay to investigate the effect of two common ALS-associated SOD1 mutations on dimerization of the SOD1 protein. Continue reading “NanoBiT Assay Applied to Study Role of SOD1 in ALS”