Browsing by Author "McCalla, Stephanie E."

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First characterization of a biphasic, switch-like DNA amplification
(2018-03) Ozay, Burcu; Robertus, Cara M.; Negri, Jackson L.; McCalla, Stephanie E.
We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1 pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.
An inexpensive, versatile, compact, programmable temperature controller and thermocycler for simultaneous analysis and visualization within a microscope
(Springer Science and Business Media LLC, 2021-05) Cruz, Pablo Martínez; Wood, Mikayla A.; Abbasi, Reha; LeFevre, Thomas B.; McCalla, Stephanie E.
Microfluidic Lab on a Chip (LOC) devices are key enabling technologies for research and industry due to their compact size, which increases the number of integrated operations while decreasing reagent use. Common operations within these devices such as chemical and biological reactions, cell growth, or kinetic measurements often require temperature control. Commercial temperature controllers are constrained by cost, complexity, size, and especially versatility for use in a broad range of applications. Small companies and research groups need temperature control systems that are more accessible, which have a wide applicability. This work describes the fabrication and validation of an inexpensive, modular, compact, and user-friendly temperature control system that functions within a microscope. This system provides precise temperature acquisition and control during imaging of any arbitrary sample which complies with the size of a microscope slide. The system includes two parts. The first part is a compact and washable Device Holder that is fabricated from high temperature resistant material and can fit securely inside a microscope stage. The second part is a robust Control Device that incorporates all the necessary components to program the temperature settings on the device and to output temperature data. The system can achieve heating and cooling times between 50°C and 100°C of 32 seconds and 101 seconds, respectively. A Bluetooth enabled smartphone application has been developed for real-time data visualization. The utility of the temperature control system was shown by monitoring rhodamine B fluorescence in a microfluidic device over a range of temperatures, and by performing a polymerase chain reaction (PCR) within a microscope. This temperature control system could potentially impact a broad scope of applications that require simultaneous imaging and temperature control.
A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions
(2021-11) Ozay, Burcu; McCalla, Stephanie E.
Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.