Browsing by Author "Negri, Jackson L."

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Decreasing the ratio non-specific to specicif miRNA amplification using a stem loop template
(Montana State University, 2017-04) Negri, Jackson L.
MicroRNA are small endogenous noncoding molecules of only 19-23 nucleotides in length. These tiny molecules play an important role in many processes in the human. Due to their limited length, miRNA sequences are very difficult to reliably detect and quantify. One possible detection method, exponential amplification reaction (EXPAR), is already being used for miRNA detection, viral DNA detection, and genomic DNA detection. The amplification process is specific and non-specific, the non-specific amplification makes it difficult to study the molecule of interest. More specific miRNA amplification would lead to a greater understanding of miRNA function in pathological processes would enable earlier disease diagnoses and potentially provide new therapies. On site diagnosis of miRNA for disease detection would be a breakthrough for biomedical research. One way to increase the specific rate of miRNA amplification is to use a stem loop template. The stem loop will help prevent the non-specific amplification of the molecule low, until the specific trigger molecule is present to initiate amplification. Altering enzyme, loop, dNTP, and magnesium concentrations were done to determine the best reaction environment. A matlab model was also created to model the reaction and determine how changing different concentrations affected the reaction.
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.