Enhancing multiplex PCR efficiency using Hot Start dNTPs

Le, Tony; Ashrafi, Elena Hidalgo; Paul, Natasha

doi:10.2144/000113298

Cited by 7 publications

(3 citation statements)

References 6 publications

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“…As stated in earlier sections, annealing temperature is an important factor for reliable PCR amplication. In conventional PCR, the annealing temperature is usually chosen between 45-65 C. 104 Using rGO, highly specic target bands were obtained at temperatures as low as 25 C and further increasing the annealing temperature did not affect the product specicity. 33 Even though experimental results showed that both GO and rGO improve the specicity of PCR, rGO provides multipleround PCR enhancement at lower concentrations in comparison to GO.…”

Section: Graphene Oxide and Reduced Graphene Oxide Assisted Polymeras...mentioning

confidence: 99%

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

et al. 2014

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Section: Graphene Oxide and Reduced Graphene Oxide Assisted Polymeras...mentioning

confidence: 99%

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

et al. 2014

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“…Improvements have been made to the storage buffers which aids in long‐term stability at −20°C and prevent decomposition of dNTPs reported during multiple freeze‐thaw cycles (Loan et al, 2019 ). Some manufacturers now offer proprietary modified dNTPs (e.g., a thermolabile 3′‐tetrahydrofuranyl protecting group on the base which is released by heat) that offer greater stability at room temperature for up to a month (Le et al, 2009 ). However, for unmodified dNTPs in standard storage buffers, manufacturers generally recommend low‐temperature storage.…”

Section: Introductionmentioning

confidence: 99%

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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“…[10][11][12] Although the use of HS polymerase results in high-specificity amplification, it significantly drives up the cost of PCR. The third approach is to employ modified primers [13][14][15][16][17] or dNTP, 18,19 and largely depends on the efficiency of the modification. [20][21][22] Recently, we presented a new QD-based nano-engineering strategy to dynamically regulate the activity of DNA polymerases and achieved a HS-like effect in conventional PCR with a high-fidelity Pfu DNA polymerase, 23,24 which was a simple and low cost HS approach without the modification in However, most of these experiments were made on an endpoint PCR assay and PCR products were analyzed by electrophoresis in agarose gels.…”

Section: Introductionmentioning

confidence: 99%

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

et al. 2015

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Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.

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Enhancing multiplex PCR efficiency using Hot Start dNTPs

Cited by 7 publications

References 6 publications

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

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

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

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