Enhancing PCR amplification and sequencing using DNA-binding proteins

Rapley, Ralph

doi:10.1007/bf02745882

Cited by 46 publications

(32 citation statements)

References 6 publications

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“…First-strand cDNA synthesis was carried out using the Superscript II reverse transcriptase (Gibco) and T7(dT)24 primer at 42°C for 1 h. T4gp32 was added to enhance the first-strand synthesis (Rapley, 1994;Nycz et al, 1998). This was followed by secondstrand synthesis using DNA polymerase I at 16°C for 2 h and incubation with T4 DNA polymerase (10 U) at 16°C for 5 min.…”

Section: Crna Probe Generation and Microarray Analysismentioning

confidence: 99%

Cellular and molecular characterization of early and late retinal stem cells/progenitors: Differential regulation of proliferation and context dependent role of Notch signaling

et al. 2004

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Retinal stem cells/progenitors that define the evolutionarily conserved early and late stages of retinal histogenesis are known to have distinct competence to give rise to stage-specific retinal cell types. However, the information regarding their innate proliferative behavior and phenotypic potential in terms of generating neurons and glia is lacking. Here we demonstrate that, like their counterparts in other central nervous system (CNS) regions during early and late stages of embryonic development, the early and late retinal stem cells/progenitors display different proliferative response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) and bias towards generating neurons or glia. Although the former predominantly generate neurons, the latter are partial towards giving rise to glia. Transcription profiling identified classes of genes that are differentially expressed in early and late retinal stem cells/progenitors in proliferating conditions and suggested that the distinct proliferative response to FGF2 and EGF is likely due to differential expression of FGF receptor 1 (FGFR1) and EGF receptor (EGFR). However, the proliferative maintenance of retinal stem cells/progenitors is likely to include other signaling pathways such as those mediated by insulin-like growth factors (IGFs) and stem cell factor (SCF). Transcription profiling of early and late retinal stem cells/progenitors in proliferating and differentiating conditions suggested a context dependent role for Notch signaling, which may constitute one of the mechanisms underlying the stage-dependent phenotypic potential of retinal stem cells/progenitors.

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Section: Crna Probe Generation and Microarray Analysismentioning

confidence: 99%

Cellular and molecular characterization of early and late retinal stem cells/progenitors: Differential regulation of proliferation and context dependent role of Notch signaling

et al. 2004

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“…There are two possible explanations for the HS effect of QDs which interacted with key components of PCR in pre-incubation. The first one is the similar mechanism of the single-stranded binding protein [10], which could selectively bind the single-stranded DNA rather than double-stranded DNA. Therefore, QDs maybe bind the primers which could block the non-specific amplifications at temperatures lower than normal PCR temperatures.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, PCR technique was frequently plagued by its low specificity and sensitivity. Many PCR enhancers have been found such as formamide [1], betaine [2], DMSO [3], gold nanoparticles [4,5], carbon nanotubes [6][7][8], carbon nanopowder [9], proteins [10][11][12], ethylene glycol/propanediol [13], dendrimers [14,15], nucleotide analogues [16] and other chemicals [17][18][19]. The low specificity and sensitivity of PCR arise partially from the non-specific amplification products, such as primer dimers and mis-priming products.…”

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

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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.

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“…In the case of soil samples, the use of a concentration of around 0.5-1 µg BSA µl -1 of PCR mix is a common procedure. Other PCR enhancements are DMSO [36] and protein T4 [37]. Given the common difficulties with optimising the reaction conditions, it is highly recommended to include a positive control.…”

Section: Pcrmentioning

confidence: 99%

A practical introduction to microbial community sequencing

Chmolowska

2013

Open Life Sciences

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The use of molecular methods is gaining popularity throughout the field of microbial community ecology studies thanks to their flexibility of application, which ranges from community structure to function and trait determination. Nonetheless, there are environmental microbiologists, who are new in the field and are just starting to get to grips with the genetic tool box. It is for them that this practitioner’s mini-review was compiled. The methods available for microbial community structure analysis are discussed, after which, the reader is introduced to sequencing, as this tool is the most appropriate and has seen the greatest development in recent years. A focus on the practical aspects of the methodology is maintained throughout. The sample preparation procedure from extraction to sequencing is described. Different applications and considerations of sequencing are briefly explained, including clone library sequencing vs. amplicon library sequencing, shotgun-metagenomics vs. metatranscriptomics and the ‘double RNA approach’.

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Enhancing PCR amplification and sequencing using DNA-binding proteins

Cited by 46 publications

References 6 publications

Cellular and molecular characterization of early and late retinal stem cells/progenitors: Differential regulation of proliferation and context dependent role of Notch signaling

Cellular and molecular characterization of early and late retinal stem cells/progenitors: Differential regulation of proliferation and context dependent role of Notch signaling

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

A practical introduction to microbial community sequencing

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