Critical Thinking Assignment Lyndon Flynn-Roach Background The experimental study, Nucleic Acid Sample Preparation Using Spontaneous Biphasic Plug Flow (Strotman et al., 2013) offers an alternative means of detecting nucleic acids through less resource-intensive and more time-efficient methods. The main objective of this study was to extract nucleic acids from a sample using a biphasic plug flow system which involves a sample and paramagnetic particles (PMPs) being placed in a capillary to purify them by moving immiscible phases. The article provides a sufficient amount of background explanation in order to explain why a newer method such as this is beneficial as well as how this method compares to more traditional tests such as polymerase chain reaction-based ones (PCR). Nucleic acid testing is often used to diagnose many types of diseases. Traditionally, PCRbased tests have been most accurate method for extracting and quantifying nucleic acids. However, the reliability of PCR-based tests depends on the accuracy of sample preparation, the quality of the nucleic acid template, and the presence of inhibitors. Additionally, PCR-based tests are very resource-intensive and involve many timeconsuming wash steps that are otherwise less practical in low resource settings that lack trained technicians and proper instrumentation. Alternative techniques capable of nucleic acid extraction are paper-based micro fluidic tests combines papers of different hydrophobic in order to filter and detect nucleic acids. While these tests are simple to perform, require little assembly, and are cost-efficient, they are however predominately used for detection of proteins in blood serum.
There are several key concepts that are essential in order to comprehend this study. Perhaps the most important concept is the usage of PCR, a technique capable of amplifying nucleic acids after they have been isolated from a sample. Another such concept in the process of aqueous layers and oil layers being disinclined to mix with one another. This creates surface tension, which can be used to create a moving force if the surface tension is uneven. A final concept to consider is adsorption which involves the adherence of the sample to glassware such as the capillary used in this study. Adsorption can frequently result in the loss of sample if surfactants are not used. In addition to concepts, there is also terminology used in this study which must be defined in order to properly understand the results. PMP s are protein-coated and polymer-coated particles that act as a solid phase during separation. They can capture a specific analyte and be permanently magnetized by an outside source in order to immobilize them (Richardson et al., 2001). Another key term is viral-like particles (VPLs) which contain viral RNA frequently found in viruses such as HIV. Methodology The initial part of the procedure involved operating the capillary in order to isolate the nucleic acids in a lysed cell sample containing PMP and lysate buffer. The biphasic plug flow technique takes advantage of the asymmetry in surface tension generated by two immiscible phases (aqueous lysate and FC-40 oil) which spontaneously pushes the sample through a capillary. The nucleic acids within the sample had bound to the PMP which were held stationary in the capillary by an external magnet. The moving oil phase expelled cellular debris as it was pushed along the capillary. The aqueous lysate phase then expelled al aqueous contaminants as it was pushed along the capillary. This
results in an isolated PMP-nucleic acid in which the nucleic acids were then unbound from the PMP by removing the magnet and then by adding elution buffer. This part of the methods addresses the initial problem with conventional nucleic acid purification procedures, which is the repetitive washing steps required for PCR-based tests. As detailed earlier, adsorption of the nucleic acids to the glass capillary walls results in the loss of product. To observe this effect, the experiment utilized two capillaries treated with red fluorescent protein capable of characterizing the amount of residual sample that adhered to the capillary walls. One trial contained a normal capillary with the residual nucleic acids, while the other trial contained a bovine serum albumin (BSA)-coated capillary with the residual nucleic acids. The results had shown that the BSA-coated capillary resulted in less adsorption and therefore less loss of nucleic acids. This method addresses the objective of the experiment by detailing how the biphasic plug flow technique is quantitatively-efficient. In traditional nucleic acid purifications, PCR can amplify the amount nucleic acids in a sample in order to obtain more conclusive results, thus effectively accounting for any loss nucleic acid sample that occurs. Since biphasic plug flow does not utilize PCR, this method helps ensure that the amount of nucleic acids purified are sufficient to for testing. The remainder of the procedure involved producing HIV VPLs by transfecting bacterial cells with plasmids and subsequently spiking the VPLs into human blood samples or fetal bovine serum in order to simulate clinical samples. The biphasic plug flow technique and cellular mrna wash purification with PCR amplification technique were both employed separated on two separate clinical samples. In order to compare the efficiency of both techniques, the RNA purified from both techniques were quantified by
constructing a standard curve using dilutions of 1.2 kb RNA standard. RNA dye was used on both clinical samples and were illuminated at 450 nm in order to capture the intensity at 540 nm. The concentration of VPLs in both clinical samples was then determined using the standard curve. The results for the fetal bovine serum sample had shown a 5- fold increase in viral RNA extraction yield compared to the conventional PCR-based methods that involved washing. The results for the human blood had simply shown a viral extraction yield of 14 viral copies per extraction but no comparison to the conventional washing-based method had been found in the study. The study made several assumptions with regards to whether certain factors could have influenced the data obtained. One of the main assumption was that the PMPs were specific to RNA, which was the nucleic acid being quantified. This assumption is not reasonable, as the study simply claims that PMP captures nucleic acids, which theoretically would also include DNA. The possibility of having DNA from the lysed sample binding to the PMPs in the capillary was not discussed in the article. A more obscure assumption is that there was no significant amount of adsorption that took place in the microtube that contained the lysed cell sample solution before it was drawn into the capillary tube. This assumption is not reasonable, as there could have been loss of nucleic acid occurring while the sample was being introduced into the capillary. Another hidden assumption is that there was no significant presence of RNA degrading compounds such as ribonucleases and oxidative chemicals within the lysed cell sample that could have affected the quality of data before drawing it into the capillary. This does not appear to be a reasonable as almost all cell types contain these types of chemicals which would almost certainly be released into the solution upon mechanically disrupting the cell.
The article outlines several arguments in order to deduce the conclusion that biphasic plug flow is an effective, low-resource approach for purifying nucleic acids. It was effectively shown that conventional washing procedures require multiple steps that are time-consuming and repetitive. The biphasic plug flow technique merely requires a single step, which effectively proves that it is more time efficient. Additionally, PCR amplification requires more expensive instrumentation coupled with skilled techniques in order to provide accurate test results, whereas the biphasic plug flow merely requires a capillary as the primary instrument. The study offers no alternative interpretations of the data since the primary objective of the experiment is mostly qualitative in nature despite the various quantitative methods used to characterize the extraction efficiency. The study had proven that the biphasic plug flow method can purify nucleic acids from a clinical sample while having extraction yields similar to the conventional wash purification method. The arguments presented in this study are quite concise and can be easily understood by a non-expert. This is due to the fact that complex experimental and arguments were often followed by a significance statement that related the relevance of that particular step to the overall scheme of the objective. An example of such an argument was the claim that BSA-treated capillaries resulted in decreased red fluorescent protein intensity. This argument was then clarified afterward in order to illustrate that BSA-treated capillaries essentially reduced the loss of red fluorescent protein by decreasing its adsorption to the capillary walls. All arguments used in the study are quite convincing, as all of the quantitative data shows that this technique can indeed be used as effectively as PCR-based tests. The main conclusion of this study was to assert that an alternative and simpler
approach toward purifying nucleic acids had been developed. The approach of the biphasic plug flow requires much few dilution steps and pipetting, which are often sources of of nucleic acid extraction yield loss when purifying by conventional methods. The study had definitively obtained an answer to its initial proposition, which was to prove that there was a more resource-efficient and simpler method to isolate nucleic acids. The study had done further quantitative analyses beyond its initial objective and also proved that the biphasic plug flow technique is capable of having extraction yields that can potentially exceed yields produced by conventional washing methods. The question of product loss through adsorption was also answered by coating all instruments with BSA in order to minimize loss. This article was significant because it gives a possible solution to the difficulty of nucleic acids testing in low resource regions where laboratories and technicians are scarce. The study helps extend knowledge about more efficient means of diagnostics for a wide range of other diseases caused by viral pathogens. It also sets a route for future studies that can take advantage of immiscible phase asymmetry in order to generate force, particular with other non-viral pathogens that need to be purified. Richardson, J., Hawkins, P., & Luxton, R. (2001). The use of coated paramagnetic particles as a physical label in a magneto-immunoassay. Biosensors and Bioelectronics, 16(9), 989-993. Thomas, P. C., Strotman, L. N., Theberge, A. B., Berthier, E., O Connell, R., Loeb, J. M.,... & Beebe, D. J. (2013). Nucleic Acid Sample Preparation Using Spontaneous Biphasic Plug Flow. Analytical chemistry, 85(18), 8641-8646.