Maximizing sensitivity and specificity of PCR by preamplification heating

D’Aquila, Richard T.; Bechtel, Lawrence; Videler, Joseph A.; Eron, Joseph J.; Gorczyca, Paul; Kaplan, Jerome I.

doi:10.1093/nar/19.13.3749

Cited by 333 publications

(102 citation statements)

References 2 publications

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“…Mispriming and primer oligomerization occur primarily before PCR amplification at room temperature, and prior to thermal cycling. Hot start PCR is commonly performed by preamplification heating in which either primers, Taq polymerase, Mg2+, or dNTPs is withheld from the reaction mixture at the initiation of PCR until the temperature becomes high enough to prevent mispriming and primer oligomerization [12,14]. The hot start PCR can conveniently be facilitated by Taqstart monoclonal antibody that neutralizes Taq polymerase activity at room temperature.…”

Section: IImentioning

confidence: 99%

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

Tani¹

1999

Acta Histochem. Cytochem

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Section: IImentioning

confidence: 99%

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

Tani¹

1999

Acta Histochem. Cytochem

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“…PCR amplification and nonradioactive SSCP (cold SSCP) were carried out to detect mutations as described previously. 14,15) The mutated SSCP bands were extracted from the gel and reamplified by PCR for 20 cycles to enrich the mutated alleles. Sequencing was performed by the dideoxy chain termination method using the AmpliTaq FS cycle-sequencing kit (Perkin-Elmer Corp., Foster City, CA).…”

Section: Methodsmentioning

confidence: 99%

Frequent p53 Gene Mutations in Soft Tissue Sarcomas Arising in Burn Scar

Nakanishi

Tomita

Yoshikawa

et al. 1999

Japanese Journal of Cancer Research

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Squamous cell carcinoma (SCC) is the commonest malignancy that arises in burn scars, which frequently contain p53 mutations. Soft tissue sarcoma (STS) also develops, though less frequently, in burn scars. p53 gene mutations were analyzed in paraffin-embedded specimens from 5 patients with STS (4 males and 1 female) that had arisen in a burn scar, by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by direct sequencing. Age at burn injury ranged from 2 to 10 (median 3) years, and STS developed with a latent period ranging from 29 to 79 (median 60) years. Histologically, all were malignant fibrous histiocytoma. The PCR-SSCP revealed aberrant bands in 4 (80%) of 5 cases. Direct sequencing revealed a total of 11 mutations in these 4 cases: 1 case had a single mutation, 1 had 2 mutations, and 2 had 4 mutations. Every tumor had at least 1 mutation that changed an amino acid, which may have provided the selection pressure for expansion. Thus, there is a high frequency of p53 gene mutations in STS appearing in burn scars. p53 mutations were also frequent in pyothorax-associated lymphoma (PAL), a lymphoma that develops in patients with long-standing pyothorax, so p53 mutations might be frequent in malignancies that develop in chronic inflammatory sites.

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“…An analogous temporary biomolecular inactivation is currently used for proteins in PCR amplification, with "hot start" polymerase enzymes. [28] In addition, the selectivity of the current acylation chemistry might enable selective control of RNA in the presence of DNA, which can otherwise be difficult to achieve. Further, since acylation blocks hybridization, it could be used to reversibly block RNA structure formation and trigger folding temporally.…”

Section: Control Of Folding Of Rna Aptamersmentioning

confidence: 99%

RNA Cloaking by Reversible Acylation

2018

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We describe a selective and mild chemical approach to controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mM) yields several 2′-OH acylations per RNA strand in as little as 10 min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity. Graphical abstractChemical switch for RNA: We describe simple selective chemical approach for control of RNA function. Polyacylation of 2′-OH groups with a nicotinyl-imidazole reagent (cloaking) switches off structure and biomolecular recognition. RNA is switched on again (uncloaking) by Staudinger reductions that remove the acyl groups. Keywordscloaking; RNA; caging; acylation; Staudinger The great complexity of RNA biology presents challenges to chemical and biological analysis. Beyond the better-studied messenger RNAs, the largest fraction of cellular RNA consists of noncoding species, including not only transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also a growing range of other long and short noncoding species, [1] including small nuclear RNAs (snRNA), [2] microRNAs (miRNA), [3] small nucleolar RNAs Correspondence to: Eric T. Kool. Supporting information for this article is given via a link at the end of the document. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript (snoRNA), [4] circular RNAs (circRNA), [5] and tRNA fragments (tRF). [6] The biological functions of a major fraction of these species remain to be characterized, and their interactions with other RNAs, proteins, and small molecules remain elusive. Recent advancements in RNA sequencing [7] and in structure mapping [8] have been important in characterizing RNAs, but the field will benefit greatly from new methods as well. For example, studies of interactions and changes that occur with RNA in time and space could benefit from methods to control structure and interactions in a chemically selective way. Moreover, the rapid rise in use of RNA as a diagnostic biomarker might also benefit from methods of controlling its properties in vitro.RNA function can be studied with methods that temporarily suspend its activity. For example, light-based blocking (photocaging) strategies have been used recently in temporal control of RNA activity. [9] In this approach, a photolabile caging group is added onto one or multiple RNA bases, [10] phosphate groups [11] or 2′-OH groups of synthetic oligoribonucleotides. [12] Upon photoirradiation, the cage is released, restoring RNA function. Such photocaging strategies have been used to control and study gene expression [10,11b,13] and ribozyme function. [12b,14] While...

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Maximizing sensitivity and specificity of PCR by preamplification heating

Cited by 333 publications

References 2 publications

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

Frequent p53 Gene Mutations in Soft Tissue Sarcomas Arising in Burn Scar

RNA Cloaking by Reversible Acylation

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