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Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions

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Chinese Chemical Letters 2022年第8期33卷 4126-4132页

作者： Junman Chen Tian Qiu michael g.mauk Zheng Su Yaguang Fan Dennis J.Yuan Qinghua Zhou Youlin Qiao Haim H.Bau Jianming Ying Jinzhao Song Key Laboratory of Clinical Laboratory Diagnostics(Ministry of Education) College of Laboratory MedicineChongqing Medical UniversityChongqing 400016China The Cancer Hospital of the University of Chinese Academy of Sciences(Zhejiang Cancer Hospital) Institute of Basic Medicine and Cancer(IBMC)Chinese Academy of SciencesHangzhou 310022China Department of Mechanical Engineering and Applied Mechanics University of PennsylvaniaPhiladelphiaPA 19104United States Department of Pathology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China Center for global Health School of Population Medicine and Public HealthChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100730China Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment Tianjin Lung Cancer InstituteTianjin Medical University General HospitalTianjin 300052China Sichuan Lung Cancer Institute Sichuan Lung Cancer CenterWest China HospitalSichuan UniversityChengdu 610041China

Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy ***,programmable endonucleases such as clustered regularly interspaced short palindromic repeats(CRISPR)associated protein(Cas)and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions,enabling their detection with deep next generation sequencing(NgS).However,the enrichment level achievable with these assays is limited by futile binding events and off-target *** overcome these shortcomings,we conceived a new assay(Programmable Enzyme-Assisted Selective Exponential Amplification,PASEA)that combines the cleavage of wild type alleles with concurrent polymerase *** PASEA increases the numbers of both wild type and mutant alleles,the numbers of mutant alleles increase at much greater rates,allowing PASEA to achieve an unprecedented level of selective enrichment of targeted *** combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification,we converted samples with0.01%somatic mutant allele fractions(MAFs)to products with 70%MAFs in a single step within 20 min,enabling inexpensive,rapid genotyping with such as Sanger ***,PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA ***-time PASEA'performance was proved equivalent to clinical amplification refractory mutation system(ARMS)-PCR and NgS when testing over hundred cancer patients'*** strategy has the potential to reduce the cost and time of cancer screening and genotyping,and to enable targeted therapies in resource-limited settings.

关键词： Mutant allele enrichment Programmable endonuclease Liquid biopsy Mutation detection Point-of-care testing CRISPR-Cas9 Recombinase polymerase amplification Nucleic acid diagnostics

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Inventing arguments / John mauk, John Metz.

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2006年

作者： John mauk John Metz.

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Tile-Drain and Denitrification Bioreactor Water Chemistry for a Soybean (<i>glycine max</i>(L.) Merr.)-Corn (<i>Zea mays</i>L.) Rotation in East-Central Missouri (USA)

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Journal of geoscience and Environment Protection 2020年第4期8卷 143-154页

作者： michael Aide Indi Braden David mauk Robert W. McAlister Byron McVay Susan Murray Samantha Siemers Sven Svenson Julie Weathers Department of Agriculture Southeast Missouri State University Cape Girardeau Missouri USA

Nitrogen transport from agriculture production fields raises the specter of environmental degradation of freshwater resources. Our objectives were to document and evaluate nitrate-N, ammonium-N, phosphorus and other nutrients emanating from a 40-ha controlled subsurface irrigation drainage technology coupled in series with a denitrification bioreactor. The intent of the denitrification bioreactor is to create an environment for anoxic microbial populations to support denitrification. We monitored the tile-drainage effluent and denitrification bioreactor water chemistry under a corn-soybean rotation to estimate the nutrient concentrations and the competence of the denitrification bioreactor to foster denitrification. Nitrate-N bearing tile drainage effluents ranged from less than 1.5 to 109 mg NO3- -N/L, with the nitrate concentration differences attributed primarily to the: 1) timing of nitrogen fertilization for corn, 2) soil mineralization and residue decomposition, and 3) intense rainfall events. The denitrification bioreactor was highly effective in reducing drainage water nitrate-N concentrations providing the rate of water flow through the denitrification bioreactor permitted sufficient time for equilibrium to be attained for the nitrate reduction reactions. The nitrate-N concentrations entering the denitrification bioreactor ranged from 0.4 to 103 mg NO3-?-N/L in 2018, whereas the outlet nitrate concentrations typically ranged from 0.3 to 5.2 mg NO3- -N/L in 2018. Nitrate tile-drainage effluent concentrations in 2019 were marginal, given soybeans obtain nitrogen from biological nitrogen fixation. Nutrient uptake by corn reduced the soil nitrate leaching pool and created nitrogen-bearing biomass, features important for formulating best management practices.