Improved PCR Flexibility with Hot Start dNTPs

Le, Tony; Paul, Natasha

doi:10.2144/000113274

Cited by 9 publications

(4 citation statements)

References 4 publications

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“…Nonspecific DNA amplification produces unexpected replicons from off-target sequences, usually causing severe issues for highly sensitive and specific target detection, − especially when millions and even billions of background DNA (bgDNA) molecules are present. , It commonly occurs in various amplification methods, including polymerase chain reaction (PCR), , loop-mediated isothermal amplification (LAMP), , and rolling circle amplification (RCA) . To avoid nonspecific amplification, the mechanism has to be clarified so that we can focus on the key points of the development of suppression methods. − However, although several challenges have been carried out, no satisfactory result has been obtained. , Usually, researchers attribute nonspecific amplification to the overlap-extension of primers, i.e., the short complementary parts at 3′-ends between two primers hybridize and extend by DNA polymerase. − Obviously, in this case, the nonspecific products should be shorter than the sum of the two primers (<40 bp). Contradictorily, most of the so-called primer dimers (nonspecific products in PCR) are longer, ranging from 50 to 150 bp. − In addition, this mechanism cannot explain that nonspecific amplification is hard to avoid even when primers are well designed …”

Section: Introductionmentioning

confidence: 99%

Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA

Song,

Hu,

Rao

et al. 2023

Anal. Chem.

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Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6−11 fully matched (9−14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer−bgDNA complex.

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Section: Introductionmentioning

confidence: 99%

Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA

Song,

Hu,

Rao

et al. 2023

Anal. Chem.

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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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“…This new dNTP analog is an elegant fusion of the secondary structure reducing nucleotide analog 7-deaza-dGTP and TriLink BioTechnologies' CleanAmp dNTP Hot Start technology. The CleanAmp dNTP technology employs a thermolabile modification group at the 3' hydroxyl that blocks dNTP incorporation at low temperatures; when released at higher thermal cycling temperatures, it produces a suitable dNTP substrate for DNA polymerase incorporation (3,4). CleanAmp 7-deaza-dGTP is described herein as an effective and simple method that greatly improves and in many cases enables specific amplification of GC-rich regions of varying complexity.…”

Section: Introductionmentioning

confidence: 99%