Nanoliter High-Throughput PCR for DNA and RNA Profiling

2009 ◽  
pp. 161-174 ◽  
Author(s):  
Colin J. H. Brenan ◽  
Douglas Roberts ◽  
James Hurley
Keyword(s):  
High Throughput ◽  
Rna Profiling ◽  
Dna And Rna

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications

PLoS ONE ◽  
2018 ◽  
Vol 13 (10) ◽  
pp. e0206085 ◽  
Author(s):  
Peter W. Inglis ◽  
Marilia de Castro R. Pappas ◽  
Lucileide V. Resende ◽  
Dario Grattapaglia
Keyword(s):  
High Throughput ◽  
Snp Genotyping ◽  
High Quality ◽  
Dna And Rna

scholarly journals Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1367
Author(s):  
Edgar Baldemar Sepúlveda-García ◽  
José Francisco Pulido-Barajas ◽  
Ariana Arlene Huerta-Heredia ◽  
Julián Mario Peña-Castro ◽  
Renyi Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Zea Mays ◽  
High Throughput ◽  
Small Rna ◽  
Abiotic Stresses ◽  
Crop Production ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Evolutionary Divergence ◽  
Rna Profiling

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

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