We’re all familiar with the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which is translated into proteins. It’s drilled into our heads from the early days of biology classes, and it’s surprisingly useful when we start exploring in our own research projects. For example, if you’re interested in gene expression, you’ll most likely be working with RNA, specifically mRNA. Messenger RNA (mRNA) is transcribed from DNA and is used by ribosomes as a “template” for a specific protein. The total mRNA in a cell represents all of the genes that are actively being transcribed. So, if you want to know whether or not a gene is being transcribed, RNA purification is a great place to start.
When preparing your RNA samples for a downstream assay, there are several roadblocks and pitfalls that could give you quite a headache. Let’s tackle two of the most common.
Antibiotic-resistant bacteria and their potential to cause epidemics with no viable treatment options have been in the news a lot. These “superbugs,” which have acquired genes giving them resistance to common and so-called “last resort” antibiotics, are a huge concern as effective treatment options dwindle. Less attention has been given to an infection that is not just impervious to antibiotics, but is actually enabled by them.
Clostridium difficile Infection (CDI) is one of the most common healthcare-associated infections and a significant global healthcare problem. Clostridium difficile (C. diff), a Gram-positive anaerobic bacterium, is the source of the infection. C. diff spores are very resilient to environmental stressors, such as pH, temperature and even antibiotics, and can be found pretty much everywhere around us, including on most of the food we eat. Ingesting the spores does not usually lead to infection inside the body without also being exposed to antibiotics.
Individuals taking antibiotics are 7-10 times more likely to acquire a CDI. Antibiotics disrupt the normal flora of the intestine, allowing C. diff to compete for resources and flourish. Once exposed to the anaerobic conditions of the human gut, these spores germinate into active cells that embed into the tissue lining the colon. The bacteria are then able to produce the toxins that can cause disease and result in severe damage, or even death. Continue reading
CRISPR is a hot topic right now, and rightly so—it is revolutionizing research that relies on editing genes. But what exactly is CRISPR? How does it work? Why is everyone so interested in using it? Today’s blog is a beginner’s guide on how CRISPR works with an overview of some new applications of this technology for those familiar with CRISPR.
Introduction to CRISPR/Cas9
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were discovered in 1987, but it took 30 years before scientists identified their function. CRISPRs are a special kind of repeating DNA sequence that bacteria have as part of their “immune” system against invading nucleic acids from viruses and other bacteria. Over time, the genetic material from these invaders can be incorporated into the bacterial genome as a CRISPR and used to target specific sequences found in foreign genomes.
CRISPRs are part of a system within a bacterium that requires a nuclease (e.g. Cas9), a single guide RNA (sgRNA) and a tracrRNA. The tracrRNA recruits Cas9, while sgRNA binds to Cas9 and guides it to the corresponding DNA sequence of the invading genome. Cas9 then cuts the DNA, creating a double-stranded break that disables its function. Bacteria use a Protospacer Adjacent Motif, or PAM, sequence near the target sequence to distinguish between self and non-self and protect their own DNA.
While this system is an effective method of protection for bacteria, CRISPR/Cas9 has been manipulated in order to perform gene editing in a lab (click here for a video about CRISPR). First, the tracrRNA and sgRNA are combined into a single molecule. Then the sequence of the guide portion of this RNA is changed to match the target sequence. Using this engineered sgRNA along with Cas9 will result in a double-stranded break (DSB) in the target DNA sequence, provided the target sequence is adjacent to a compatible PAM sequence. Continue reading
Yersinia pestis. See page for author [Public domain], via Wikimedia Commons
While scientists using ancient DNA analysis are learning how Yersinia pestis
developed over time into the causative agent of three worldwide pandemics, there is still much to learn about the early hours and days of an organism infected with the plague. Y. pestis
still infects humans so any insight into disease progression is useful for determining treatment timing and even developing novel treatments to supplement or replace antibiotics. A 2012 study
observed how Y. pestis
injected into mice spread throughout the body using bioluminescent imaging to track the infection. More recent research reported in PLOS ONE
used a more real-world route of infection by introducing an aerosolized Y. pestis
to a nonhuman primate model and tracking the transcripts altered during the first 42 hours of infection. Continue reading
Figure 5 shows typical scanned images of bead-array for analyzing adjacent normal tissue and tumor tissue. Huang et al. (2015) Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array. PLOS ONE 10(4):e0123420. doi:10.1371/journal.pone.0123420.g005
The ideal cancer detection method would involve giving a sample of blood or tissue and using DNA or RNA analysis to determine if there were any gene sequence or gene expression changes that are known hallmarks of cancer. Unfortunately, most current screening methods used are not so precise and in some cases are invasive. However useful tests for colon cancer may be, many people do not subject themselves to the standard colonoscopy. What if there was an easier, noninvasive method that could be used to screen for cancer and detect changes at the early, easily treatable stages of cancer? A recent article in PLOS ONE
describes just such a mutation detection method for colorectal cancer using purified nucleic acid with a method that involves emulsion PCR, bead arrays and fluorescent probes. Continue reading
Riboprobes are RNA probes that can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
Viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Transcription of RNA is performed with the appropriate RNA polymerase (T3, T7 or SP6), depending on the RNA polymerase promoter sites present in the chosen vector. Because these polymerases are extremely promoter-specific (i.e., there is almost no transcriptional cross talk), virtually homogeneous RNA can be obtained using plasmid DNA as the template in a transcription reaction. When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an appropriate restriction site before the transcription reaction and only discrete “run-off” transcripts are obtained, virtually free of vector sequences. RNA transcripts may be used to generate radioactive probes for hybridization to Northern and Southern blots, plaque and colony lifts as well as non-radioactive probes (i.e, labeled with digoxgenin)for in situ hybridization.
Recent references using riboprobes include: Continue reading
It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those unfamiliar or new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.
Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest. Continue reading
Protein location: outer mitochondrial membrane (Yeast in vitro import assay)
Curado, S. et.al. (2010) Dis.Mod. Mech. 3, 486-95. PubMed ID 20483998.
Chemically mutagenized zebra fish were assayed for liver defects in their F3 progeny.This screen led to the identification of mutant called oliver. Oliver mutants have an o-shaped liver of a much deprived size due to the depletion of most of the hepatocytes. This mutation maps to the Tomm22gene which encodes a translocase of the outer membrane and thought to play an important role in protein import into mitochondria. Various Tomm22 mutants were expressed and used in a yeast in vitro import systemto determine if correct inserted into the yeast outer mitochondrial membrane.
Protein modification: hydroxylation
Serchov, T. et.al. (2010) J. Biol. Chem. 285, 21223-232. PubMed ID 20418372 .
Proline hydroxylation is also a vital component of hypoxia via hyposxia inducible factors. The cellular response to hypoxia involves the induction of the hypoxia-inducible factor considered to be the major transcription factor involved in gene regulation of hypoxia. This factor is hydroxylated by prolyl-hydroxase dolman proteins (PHDs). To investigate if a newly identified component of the hypoxia pathway (Elk3) is also hydroxylated, proteins were expressed +/- PHDs cofactors and protein mobility was measured via gel analysis.
Gene Experession: Programmed Ribosomal Frameshift
Kobayashi, Y. et.al. (2010) J. Biol. Chem. 285, 19776-784. PubMed ID 20427288.
Programmed -1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses (HIV-1 for example). Recently a genome-wide screen demonstrated that down regulation of eukaryotic release factor (eRF1) inhibited HIV-1 replication. In order to characterize the dose dependent effect of eRF1, increasing amounts were expressed in the presence of dual luciferase reporter vectors harboring a HIV-1 PRF signal
The plans had been made, details finalized and all expenses paid. I was to travel to the south coast of England to complete my training for the British Sub-Aqua Club Sports Diver certificate. I boarded a train from London’s Waterloo station down to the quiet seaside resort of Bournemouth where I was received by relatives. For the next two weeks I commuted to the nearby harbor town of Poole and headed out on a rigid hull inflatable boat with five other students to complete a series of required dives. The testosterone-induced camaraderie soon brought us together into a close-knit group. We were assigned our respective diving ‘buddies’- a practice that is almost a mandatory requirement of amateur sport diving. We quickly picked up on the diving lingo and were Hi-fiving our way to the end of each day.
All of our sorties out to sea went according to plan. That is, until the final afternoon. As we were heading back to the safety of the mooring station the weather took a turn for the worst. Surging waves reduced visibility to little more than a few feet and with the quickly darkening skies we knew we were in trouble. In desperation the pilot of the boat radioed for help. Minutes later we were spotted by the coastal ‘cavalry guard’- a British Navy Sea King helicopter equipped with all the fittings that one might expect for a major rescue operation. Fortunately the terrifying experience of being stranded out at sea ended without further incident. We were escorted to the calmer waters of a local bay from which we headed home for a feast of fried fish served in greasy, vinegar-sodden newspaper (the quintessentially English supper). That same evening we all reconvened to mull over the events as they had unfolded. We bonded socially knowing that, in the midst of our differences, there was at least one thread of commonality by which we could all relate to each other. We were all now sports divers with a story to tell.
A craving for social connection is a deeply-rooted aspect of the human psyche (1). Continue reading
How do you explain the phenomenon of incomplete penetrance, which happens when individuals carrying an allele for a given phenotype don’t always express the phenotype? For instance, individuals carrying the same mutation associated with a genetic disease do not always develop that disease.
Sometimes environment influences gene expression and plays a role, or other genetic differences among the individuals of a population can affect the expression of the gene in question. But, incomplete penetrance is also observed in model organisms that are raised in controlled environmental conditions and that have “identical” genetic makeup.
Biologists have proposed that random variability in gene expression could account for such events, and in clonal populations of microorganisms random variation in gene expression may even be important for generating genetic variability. However, in more complex organisms that have specific cell types organized into tissues and organs, gene expression needs to be highly controlled for the organism to develop properly. So, if there are random fluctuations in gene expression, somehow they need to be “buffered” in normal development.
Until the recent Nature paper published by Raj et al. (1), little experimental data existed to support the theory that essential developmental pathways include mechanisms to buffer the effects of random variations in gene expression. Continue reading