Dataset Information

Generating single-stranded DNA circles with minimal resources.

ABSTRACT: Single-stranded circular oligonucleotides are heavily utilized in rolling circle amplification and rolling circle transcription technologies. Although various reported methodologies are available to synthesize circular, single-stranded DNA (ssDNA), the unduly complicated protocols and the associated cost minimize the utility of these methodologies to a non-expert or a beginner in the field. Our protocol provides the simplest yet robust synthesis of circular ssDNA templates to be utilized in various applications, using minimal resources.•In this manuscript, we describe the most basic approach to synthesize circular ssDNA.•Our method utilizes the minimal resources, yet it is robust.•The utility of the methodology is very high for a non-expert or a beginner in the field.

SUBMITTER: Ford A

PROVIDER: S-EPMC8374276 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Generating single-stranded DNA circles with minimal resources.

Ford Amanda A Miller LaShawna L Trant Jordan J Nawarathne Irosha N IN

MethodsX 20210308

Single-stranded circular oligonucleotides are heavily utilized in rolling circle amplification and rolling circle transcription technologies. Although various reported methodologies are available to synthesize circular, single-stranded DNA (ssDNA), the unduly complicated protocols and the associated cost minimize the utility of these methodologies to a non-expert or a beginner in the field. Our protocol provides the simplest yet robust synthesis of circular ssDNA templates to be utilized in vari ...[more]

PMID: 34434820

Similar Datasets

Project description:UnlabelledNeisseria gonorrhoeae is naturally competent for transformation. The first step of the transformation process is the uptake of DNA from the environment into the cell. This transport step is driven by a powerful molecular machine. Here, we addressed the question whether this machine imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at similar rates. The fluorescence signal associated with the uptake of short DNA fragments labeled with a single fluorescent marker molecule was quantified. We found that ssDNA with a double-stranded DNA uptake sequence (DUS) was taken up with a similar efficiency as dsDNA. Imported ssDNA was degraded rapidly, and the thermonuclease Nuc was required for degradation. In a nuc deletion background, dsDNA and ssDNA with a double-stranded DUS were imported and used as the substrates for transformation, whereas the import and transformation efficiencies of ssDNA with single-stranded DUS were below the detection limits. We conclude that the DNA uptake machine requires a double-stranded DUS for efficient DNA recognition and transports ssDNA and dsDNA with comparable efficiencies.ImportanceBacterial transformation enables bacteria to exchange genetic information. It can speed up adaptive evolution and enhances the potential of DNA repair. The transport of DNA through the outer membrane is the first step of transformation in Gram-negative species. It is driven by a powerful molecular machine whose mechanism remains elusive. Here, we show for Neisseria gonorrhoeae that the machine transports single- and double-stranded DNA at comparable rates, provided that the species-specific DNA uptake sequence is double stranded. Moreover, we found that single-stranded DNA taken up into the periplasm is rapidly degraded by the thermonuclease Nuc. We conclude that the secondary structure of transforming DNA is important for the recognition of self DNA but not for the process of transport through the outer membrane.

Dataset Information

Generating single-stranded DNA circles with minimal resources.

Publications

Generating single-stranded DNA circles with minimal resources.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets