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A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

ABSTRACT:

Key message

In-house production of a positive selection cloning vector could be simple, efficient and low cost.

Abstract

DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%.

Supplementary information

The online version contains supplementary material available at 10.1007/s13205-022-03289-x.

SUBMITTER: Nawawi O

PROVIDER: S-EPMC9363543 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Nawawi Omar O Abdullah Mohd Puad MP Yusuf Chong Yu Lok CYL

3 Biotech 20220809 9

<h4>Key message</h4>In-house production of a positive selection cloning vector could be simple, efficient and low cost.<h4>Abstract</h4>DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common ...[more]

PMID: 35965659

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Project description:IntroductionCytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world.ObjectiveThis study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test.Study designThe methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n=30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study.ResultsThe in-house assay had a limit of detection and quantification of 60.3IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively.ConclusionsThese results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Dataset Information

A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Key message

Abstract

Supplementary information

Publications

A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Similar Datasets

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