Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore

Craig, Jonathan M.; Laszlo, Andrew H.; Derrington, Ian M.; Ross, Benjamin; Brinkerhoff, Henry; Nova, Ian C.; Doering, Kenji; Tickman, Benjamin I.; Svet, Mark T.; Gundlach, Jens H.

doi:10.1371/journal.pone.0143253

Cited by 28 publications

(28 citation statements)

References 13 publications

(33 reference statements)

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“…In order to have single-nucleotide sensitivity, it is key that the size of the nanopore (diameter and length) is commensurate with the dimensions of the DNA bases [ 5 , 16 ]. Biological pores such as aHL [ 1 , 2 , 4 , 19 – 21 ] or MspA [ 5 – 7 , 22 – 27 ], with a geometry similar to the size of ssDNA nucleotides [ 5 ], have been used to study DNA.…”

Section: Introductionmentioning

confidence: 99%

MspA nanopore as a single-molecule tool: From sequencing to SPRNT

Laszlo

Derrington

Gundlach

2016

Methods

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105

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Single-molecule picometer resolution nanopore tweezers (SPRNT) is a new tool for analyzing the motion of nucleic acids through molecular motors. With SPRNT, individual enzymatic motions along DNA as small as 40 pm can be resolved on sub-millisecond time scales. Additionally, SPRNT reveals an enzyme’s exact location with respect to a DNA strand’s nucleotide sequence, enabling identification of sequence-specific behaviors. SPRNT is enabled by a mutant version of the biological nanopore formed by Mycobacterium smegmatis porin A (MspA). SPRNT is strongly rooted in nanopore sequencing and therefore requires a solid understanding of basic principles of nanopore sequencing. Furthermore, SPRNT shares tools developed for nanopore sequencing and extends them to analysis of single-molecule kinetics. As such, this review begins with a brief history of our work developing the nanopore MspA for nanopore sequencing. We then describe the underlying principles of SPRNT, how it works in detail, and propose some potential future uses. We close with a comparison of SPRNT to other techniques and we present the methods that will enable others to use SPRNT.

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Section: Introductionmentioning

confidence: 99%

MspA nanopore as a single-molecule tool: From sequencing to SPRNT

Laszlo

Derrington

Gundlach

2016

Methods

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105

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“…2A) would be conducted on a Sanger sequencer (Fig. 2B Step VI and Table 1); however, d5SCIS and dNaM were recently sequenced using the MspA protein nanopore (Craig et al, 2015). This opens the possibility of sequencing these unnatural nucleotides by Oxford Nanopore’s MinION high-throughput NGS platform that uses a protein nanopore (Jain et al, 2015); however, this has yet to be achieved.…”

Section: Comparison and Contrast Of The Three Og Sequencing Methodsmentioning

confidence: 99%

“…The concept of sequencing d5SICS and dNaM with the MspA protein nanopore has been demonstrated (Craig et al, 2015). …”

Section: Figmentioning

confidence: 99%

Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine

Fleming

Ding

Burrows

2017

Methods in Enzymology

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The DNA base guanine (G) can be oxidatively modified to 8-oxo-7,8-dihydroguanine (OG). Extraction of genomic DNA followed by nuclease digestion and mass spectrometry analysis have found OG is present at background levels of ~1 out of 106 Gs; however, this approach cannot determine the locations for the OGs in the genome. Thus, in this methods report, we outline three different methods (A, B, and C) for sequencing OG in DNA. Method A sequences OG by utilizing the base excision repair pathway to delete the OG nucleotide from the DNA that is then detected by Sanger sequencing as a deletion signature. Method B sequences OG by harnessing the base excision repair pathway to convert OG to an unnatural DNA base pair followed by Sanger sequencing to locate the unnatural base pair indicating where OG was located. Method C (i.e., OG-Seq) takes genomic DNA sheared to ~150 bps followed by selectively biotinylating the OG-containing fragments for affinity purification and enrichment of the OG-modified strands. The OG-modified fragments are sequenced on a next-generation sequencing platform to locate OG on the genomic scale with a resolution of ~150 bps. The methods outlined are then compared and contrasted allowing researchers to select the one that best suits their experimental goals.

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“…156 Moreover, detection of unnatural nucleotides dNaM and d5SICS has been enabled by the MspA–phi29 system. 121 …”

Section: Biological Nanoporesmentioning

confidence: 99%