Fig 6 - available via license: Creative Commons Attribution 4.0 International
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Chromatograms of the sequencing library fragment sizing analysis carried out on an Agilent Bioanalyzer 2100. WPA: whole circular plasmid amplification, OverFlapWPA: OverFlapPCR-based whole plasmid amplification, and OverFlapAsymWPA: OverFlapPCR based asymmetric whole plasmid amplification. The 35 bp (green) and 10 380 bp (purple) peaks represent the upper and lower markers employed by data analysis software for the internal calibration of each analysis. https://doi.org/10.1371/journal.pone.0262968.g006
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Over the decades, practical biotechnology researchers have aimed to improve naturally occurring proteins and create novel ones. It is widely recognized that coupling protein sequence randomization with various effect screening methodologies is one of the most powerful techniques for quickly, efficiently, and purposefully acquiring these desired imp...
Contexts in source publication
Context 1
... results revealed that, contrary to our expectations, products of various sizes formed our library. This indicated that some deletions and duplications occurred (Fig 6 WPA) in addition to the randomization of the target sequence. Subsequently, the acquired sequencing data partially confirmed this suspicion because many randomized translated sequences without a template bias were shorter than 18 aa (54 nucleotides). ...
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... PCR -a reliable approach for generation of random sequence plasmid DNA libraries Therefore, we proceeded with generating a sequencing library and assessing its quality. Data from BioAnalyzer revealed that the proportion of shorter and longer fragments was significantly decreased, and the single peak of the 273 bp target could be identified (Fig 6 OverFlapWPA). NGS analysis revealed that, compared to traditional WPA, the number of unique protein CDSs increased more than four times, reaching 4 636. ...
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... the agarose gel electrophoresis and capillary electrophoresis-based DNA fragment analysis results were peculiar. They revealed that the size of the library's major PCR product was 492 bp, while the amount of expected 273 bp fragments, although shifted by 5 bp, was significantly lower (Fig 6 OverFlapAsymWPA). Therefore, we decided to perform an additional purification with size selection beads to remove any DNA fragments larger than 300 bp. ...
