Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

Lebedev, A.; Paul, Natasha; Yee, Joyclyn; Timoshchuk, V. A.; Shum, Jonathan; Miyagi, Kazuya; Kellum, Jack; Hogrefe, Richard I.; Zon, Gerald

doi:10.1093/nar/gkn575

Cited by 45 publications

(53 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, most suppliers of Taq-polymerase offer the native (nonrecombinant) enzyme at only slightly higher prices. A minor disadvantage might be that advanced features engineered in the recombinant Taq-polymerase, like the activation by heat exposure in order to avoid premature enzyme activity, are not available for the native enzymes (Kellogg et al 1994, Lebedev et al 2008). …”

Section: Selection At Gene or Phenotypic Levelmentioning

confidence: 99%

The role of molecular markers and marker assisted selection in breeding for organic agriculture

et al. 2010

View full text Add to dashboard Cite

Plant geneticists consider molecular marker assisted selection a useful additional tool in plant breeding programs to make selection more efficient. Standards for organic agriculture do not exclude the use of molecular markers as such, however for the organic sector the appropriateness of molecular markers is not self-evident and is often debated. Organic and low-input farming conditions require breeding for robust and flexible varieties, which may be hampered by too much focus on the molecular level. Pros and contras for application of molecular markers in breeding for organic agriculture was the topic of a recent European plant breeding workshop. The participants evaluated strengths, weaknesses, opportunities, and threats of the use of molecular markers and we formalized their inputs into breeder's perspectives and perspectives seen from the organic sector's standpoint. Clear strengths were identified, e.g. better knowledge about gene pool of breeding material, more efficient introgression of new resistance genes from wild relatives and testing pyramided genes. There were also common concerns among breeders aiming at breeding for organic and/ or conventional agriculture, such as the increasing competition and cost investments to get access to marker technology, and the need for bridging the gap between phenotyping and genotyping especially with complex and quantitative inherited traits such as nutrient-efficiency. A major conclusion of the authors is that more interaction and mutual understanding between organic and molecular oriented breeders is necessary and can benefit both research communities.

show abstract

Section: Selection At Gene or Phenotypic Levelmentioning

confidence: 99%

The role of molecular markers and marker assisted selection in breeding for organic agriculture

et al. 2010

View full text Add to dashboard Cite

show abstract

“…Although the use of HS polymerase results in high-specificity amplification, it has greatly increased the cost of PCR amplification. The third approach is to employ modified primers [20][21][22][23] or dNTP [24], and largely depends on the efficiency of the modification [25][26][27].…”

Section: Introductionmentioning

confidence: 99%

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

Sang¹,

Yang²,

Lin³

et al. 2014

ABBS

View full text Add to dashboard Cite

Quantum dots (QDs) are of great interest due to their unique chemical and physical properties. Recently, a hot start (HS) polymerase chain reaction (PCR) amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high-fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was preincubated for 2 h at 508 8 8 8 8C. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incubation at 508 8 8 8 8C before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA polymerases.

show abstract

“…Although capable of significantly suppressing non-specific amplification, these polymerase-based approaches have significantly added the cost for PCR amplification. The third approach achieves HS effects by the modifications of the primers [29][30][31][32] or dNTP [33], and such methods largely depend on the efficiency of the modification of primers or dNTP.…”

Section: Introductionmentioning

confidence: 99%

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

Sang

Yang

Wang

et al. 2013

Journal of Experimental Nanoscience

View full text Add to dashboard Cite

Hot-start (HS) effects were investigated in pfu-based polymerase chain reaction (PCR), when water-soluble CdTe quantum dots (QDs) were introduced in the PCR system. The HS effects were demonstrated by the higher amplicon yields and excellent suppression of non-specific amplification after pre-incubation of PCR mix with QDs between 35 C and 56 C. DNA targets were well amplified even after PCR mixture was pre-incubated 1 h at 50 C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the pfu polymerase concentration, suggesting that there was an interaction between QDs and pfu DNA polymerase. Moreover, control experiment indicated that HS effect is not primarily due to the reduced pfu polymerase concentration resulted from the above interaction. Fluorescence correlation spectroscopy (FCS), a single molecule detection method, was used to investigate the possible mechanism of HS PCR with QDs. Preliminary FCS results suggested that CdTe QDs may directly interact with pfu DNA polymerase, rather than other components in the PCR system. Furthermore, results demonstrated that the interaction between QDs and pfu resulted in a reduction in pfu polymerase concentration. This study provided a good start to investigate potential implications of QDs in other key molecular biology techniques.

show abstract

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

Cited by 45 publications

References 80 publications

The role of molecular markers and marker assisted selection in breeding for organic agriculture

The role of molecular markers and marker assisted selection in breeding for organic agriculture

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

Contact Info

Product

Resources

About