Improved PCR specificity with Hot Start PCR primers

Ashrafi, Elena Hidalgo; Paul, Natasha

doi:10.2144/000113240

Cited by 10 publications

(5 citation statements)

References 4 publications

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“…Long artifacts comprise off-target products which contain additional sequences that do not or only partially overlap with the targeted sequence [5] . Although some papers describe how to prevent amplification of nonspecific products [6] , [7] , [8] , hardly any paper deals with the conditions leading to these off-target products.…”

Section: Introductionmentioning

confidence: 99%

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Ruiz‐Villalba

Pelt-Verkuil

Gunst

et al. 2017

Biomolecular Detection and Quantification

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Section: Introductionmentioning

confidence: 99%

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Ruiz‐Villalba

Pelt-Verkuil

Gunst

et al. 2017

Biomolecular Detection and Quantification

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“…For example, primers containing the temperature-sensitive 4-oxo-tetra-decyl (OXT) phosphotriester modification demonstrated superior performance compared to unmodified PCR primers in their ability to amplify four targets from human total RNA both separately and simultaneously in a one-step reverse-transcription PCR [55]. Still, the probability to find an optimal set of primers able to target all templates in a mixed sample is very low.…”

Section: Factors That Can Diminish Artifacts Formation In Multi-templmentioning

confidence: 99%

Multi-template polymerase chain reaction

Kalle

Kubista

Rensing

2014

Biomolecular Detection and Quantification

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PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

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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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Improved PCR specificity with Hot Start PCR primers

Cited by 10 publications

References 4 publications

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Multi-template polymerase chain reaction

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

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