TbEu(BTC) as a dNTP label for LAMP outcome in nucleic acid testing

Gao, Chenfeng; Wong, Joseph Jo Yin; Hall, Elizabeth A. H.

doi:10.1002/elan.202200504

Cited by 1 publication

(1 citation statement)

References 32 publications

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“…However, for unmodified dNTPs in standard storage buffers, manufacturers generally recommend low‐temperature storage. In general, dNTPs are found to have time‐limited stability at ambient temperature and even at 4°C, whereas at higher temperatures the degradation rate increases: from other work from our group, this is significant above 60°C, in which a 10 mM solution of each dNTP (dATP, dCTP, dGTP, and dTTP) in amplification buffer has shown circa 7% per hour degradation (Gao et al, 2023 ). Thus, in low‐resource contexts where reliable consistent cold temperature storage is not always available, for example, due to power cuts, a method to produce dNTPs on‐demand can become critical.…”

Section: Introductionmentioning

confidence: 78%

Biocatalytic synthesis of 2′‐deoxynucleotide 5′‐triphosphates from bacterial genomic DNA: Proof of principle

Bird

Molloy

Hall

2023

Biotech & Bioengineering

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2′-deoxynucleoside 5′-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR).These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in lowand middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solidstate bioreactor for on-demand dNTP production.

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Section: Introductionmentioning

confidence: 78%