Robust PCR Amplification of GC-Rich Targets with Hot Start 7-deaza-dGTP

Shore, Sabrina; Paul, Natasha

doi:10.2144/000113552

Cited by 11 publications

(7 citation statements)

References 6 publications

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“…The Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits primers used for amplification of the human BRAF gene were adopted from the literature [27]. The specificity and sequence features of the used primers (table 1) were checked using primerblast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and oligoanalyzer online tools (https://sg.idtdna.com/calc/analyzer), respectively.…”

Section: Primer Selectionmentioning

confidence: 99%

Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits

2019

Biointerface Res Appl Chem

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Various strategies have been suggested for successful amplification of the hard-to-amplify nucleic acids such as the inclusion of various chemical or biological materials in the in vitro nucleic acid amplification reactions, particularly polymerase chain reaction (PCR), and adjustment of the cycling programs. Although much efforts have been madefor improvements in the enhancement of polymerase chain reactions of high GC content nucleic acids, still significant challenges remain. In this study, the effects of citrate-coated AuNP and chamomile extract-coated AuNP (p-AuNP) on amplification of three genes with significant different GC percentage were evaluated. Owing to the enormous potential of gold nanoparticles in the enhancement of the PCR reactions, we showed the promising and consistent findings on the application of very dilute biocompatible chamomile-gold nanoparticles as safe and low-cost nanomaterials for molecular amplification of GC-rich DNA samples. We hypothesized that green AuNPs, which have different surface chemistry from cit-AuNPs, not only do not interfere with the PCR reactants but also are capable of enhancing PCR reactions. These results, for the first time, confirm the potential of using the green gold nanoparticles in the heat-assisted enzymatic in vitro reactions, suggesting Chamomile gold nanoparticles as reliable component of any PCR kits.

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Section: Primer Selectionmentioning

confidence: 99%

Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits

2019

Biointerface Res Appl Chem

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“…GC rich regions produce complex inter and intra strand folding (hairpins and loops) due to increased hydrogen bonding with neighbouring cytosine and guanine (Musso et al, 2006). These secondary structures in DNA are resistant to melting and cause Taq DNA polymerases to stall and they also hampers primer annealing, resulting in incomplete or non-specific amplification (Shore and Paul, 2010;Hubé et al, 2005). As a result of this, several approaches have been tried to amplify GC rich templates by adding dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, formamide, betaine, 7-deaza-dGTP, and dUTP in the reaction mixture (Baskaran et al, 1996;Chakrabarti and Schutt, 2001;Henke et al, 1997;Kang et al, …”

Section: Introductionmentioning

confidence: 99%

PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA

Sharma¹

2015

Adv. Anim. Vet. Sci.

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| Amplification of GC rich templates using PCR is usually difficult compared to non-GCrich targets. GC rich regions produce complex inter and intra strand folding (hairpins and loops) due to higher hydrogen bonding with neighbouring cytosine and guanine. These secondary structures in DNA are resistant to melting and cause Taq DNA polymerases to stall; they also hamper primer annealing, resulting in incomplete or non-specific amplifications. The aim of this study was to develop an easy PCR protocol to amplify very high GC rich (88% in the coding region) protamine gene of chicken. The cDNA used for amplification was synthesised from testis RNA and PCR products were detected by agarose gel electrophoresis. Protamine amplicon successfully amplified only with Go Taq DNA polymerase in the presence of DMSO. Optimization of MgCl 2 concentration, buffer strength, annealing temperature, DMSO and the DNA template concentration, were important in the PCR reaction resulting in successful amplification. In addition, PCR started directly at hot plate is also important to get rid of primer-dimer formation.

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“…[1] These secondary structures in DNA are resistant to melting and cause Taq DNA polymerases to stall and also hampers primer annealing, resulting in incomplete or non-specific amplification. [23]…”

Section: Introductionmentioning

confidence: 99%

“…These include addition of organic substances (additives), use of modified dNTPs and modification of thermal cycling program in PCR. [2] The additives improve the amplification by unwinding the double stranded DNA (dsDNA) helix and thereby reducing the melting temperature. The most prominent PCR enhancing additives currently used are either betaine, small sulfoxides such as dimethyl sulfoxide (DMSO), formamide or reducing compounds such as beta-mercaptoethanol or dithiothreitol.…”

Section: Introductionmentioning

confidence: 99%

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA

2013

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CONTEXT:Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters.AIM:Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.SETTINGS AND DESIGN:Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells.MATERIALS AND METHODS:A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase.RESULTS:Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used.CONCLUSIONS:It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.

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Robust PCR Amplification of GC-Rich Targets with Hot Start 7-deaza-dGTP

Cited by 11 publications

References 6 publications

Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits

Synthesis, characterisation, and application of chamomile gold nanoparticles in molecular diagnostics: a new component for PCR kits

PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA

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