Title
A low-cost and open-source protocol to produce key enzymes for molecular detection assays
Date Issued
17 December 2021
Access level
open access
Resource Type
journal article
Author(s)
Mendoza-Rojas G.
Sanchez-Castro A.
Tello L.
Cabrera-Sosa L.
Nakamoto J.A.
Peñaranda K.
Publisher(s)
Cell Press
Abstract
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).
Volume
2
Issue
4
Language
English
OCDE Knowledge area
Bioquímica, Biología molecular
Subjects
Scopus EID
2-s2.0-85118479458
PubMed ID
Source
STAR Protocols
ISSN of the container
26661667
Source funding
Sponsor(s)
We are very thankful to Dr. Marcos Milla for donating equipment that was used in this study and others. We would also like to thank all lab members of the Adaui and Milón groups for their help, support, and great working atmosphere. This work was supported by grants from the Peruvian Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica [ 070-2020-FONDECYT ] to VA and [ 036-2019-FONDECYT-BM-INC.INV ] and [ 154-2017-FONDECYT ] to PM.
We are very thankful to Dr. Marcos Milla for donating equipment that was used in this study and others. We would also like to thank all lab members of the Adaui and Mil?n groups for their help, support, and great working atmosphere. This work was supported by grants from the Peruvian Fondo Nacional de Desarrollo Cient?fico, Tecnol?gico y de Innovaci?n Tecnol?gica [070-2020-FONDECYT] to VA and [036-2019-FONDECYT-BM-INC.INV] and [154-2017-FONDECYT] to PM. Conceptualization and methodology, R.A. K.P. J.A.N. L.C.-S. and P.M.; investigation, G.M.-R. A.S.-C. V.S.-V. L.T. K.P. and R.A.; formal analysis, R.A. K.P. and P.M. writing ? original draft, V.S.-V, A.S.-C. K.P. R.A. and P.M. writing ? review & editing, V.S.-V. A.S.-C. G.M.-R. R.A. L.T. K.P. J.A.N. L.C.-S. V.A. and P.M.; visualization, V.S.-V. and A.S.-C.; supervision, R.A. and P.M.; project administration, P.M. and K.P. The authors declare no competing interests.
Sources of information:
Directorio de Producción Científica
Scopus