A General Method for the Isolation of Rna Complementary to Dna

Bolton, Ellis T.; McCarthy, Brian J.

doi:10.1073/pnas.48.8.1390

Cited by 266 publications

(73 citation statements)

References 14 publications

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“…Filters were washed twice for 5 min at 50°C-60°C in 1 x SSC, 0.1% SDS and exposed for several days with two intensifying screens. To control the stringency, we estimated the melting temperature T, for oligonucleotide probes from the following equation: T, = 81.5"C + 16.6 logro[Na+] + 0.41(%G + C) -O.b3(%formamide) -6001 I, where I = length of the hybrid in base pairs (Bolton and McCarthy, 1962). Conditions for our oligonucleotide probes were 18OC below T, for hybridization and 18OC to 28OC below T, for washing.…”

Section: Camp-dependentmentioning

confidence: 99%

The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-Dependent protein kinase

Boshart

Weih

Nichols

et al. 1991

Cell

180

100

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Section: Camp-dependentmentioning

confidence: 99%

The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-Dependent protein kinase

Boshart

Weih

Nichols

et al. 1991

Cell

180

100

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“…The melting temperature for mixed bases is calculated by averaging nearest neighbor thermodynamic parameters-enthalpy and entropy values-at each mixed site; extinction coefficient is similarly predicted by averaging nearest neighbor values at mixed sites [2,3]. Mismatched pairs can be taken into account since the parameters provide for DNA/DNA duplexes and dangling ends, Nearest neighbor thermodynamic parameters according to SantaLucia [13] b Ambiguity codes c Sequence linguistic complexity measurement calculated using the alphabet-capacity L-gram method which are unmatched terminal nucleotides [14][15][16]. The melting temperature for primer (probe) self-or cross-dimers and for in silico PCR experiments with oligonucleotides having mismatches to the target is calculated using values for the thermodynamic parameters for a nucleic acid duplex.…”

Section: Pcr Primer Design Generalitiesmentioning

confidence: 99%

“…The two equations above assume that the stabilizing effects of cations are the same on all base pairs. The melting temperature of the PCR product is calculated using the formula [15]:…”

Section: Melting Temperature Calculationmentioning

confidence: 99%

FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis

Kalendar

Lee

Schulman

2013

Methods in Molecular Biology

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This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a program to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, and linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with CG or AT skew, of CG content and purine-pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyzes sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at

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“…Although immobilized DNA techniques for nucleic acid hybridization (1,2) have been extensively used to measure nucleotide sequence homology between nucleic acid molecules, they are not suitable for RNA-RNA hybridizations or for RNA-DNA hybridizations that use radioactive DNA. Such hybridization experiments would be greatly facilitated by immobilization of RNA on a solid support.…”

mentioning

confidence: 99%

Nucleic Acid Hybridization with RNA Immobilized on Filter Paper

Saxinger

Ponnamperuma

Gillespie

1972

Proc. Natl. Acad. Sci. U.S.A.

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(3)(4)(5)(6)(7)(8), and the techniques have been useful in some circumstances.In the method we present, RNA is covalently bonded to cellulose with the aid of the condensing agent carbonyldiimidazole (COIm2) in an uninterrupted one-step reaction, or in a two-step reaction wherein the process is interrupted between the formation of an activated phosphate intermediate and its subsequent reaction with RNA. COIm2 is neither toxic nor explosive, so that its application in these reactions requires no special apparatus and may proceed completely unattended. The simplicity, convenience, and versatility of this method recommends its use in routine or repetitive microanalytical scale experiments on filter discs, as well as in preparative-scale work with powder. In the two-step technique, the attachment yields with several RNA species were nearly quantitative and, in the one case tested-poly(A)- For attachment in the one-step reaction, these RNA preparations were converted to their tributylammonium salt by dialysis against 50 mM Bu3N * HCl (pH 6) or by passage through a Biogel P2 column equilibrated with 5 mM Bu3N* HCl (pH 6).Preparation of RNA Filters (One-Step Reaction). RNA was spotted on 7-mm (diameter) paper discs air-dried for 10 min, then. dried overnight under reduced pressure over P205 and KOH. During the subsequent bonding of RNA to filter paper, all operations were done in a dry atmosphere or in sealed vials. Organic solvent containing condensing agent (100 mg/ml, unless specific otherwise) was delivered to a vial containing replicate RNA-coated filters so that all of the filters were immersed. There is a slight but detectable transfer of RNA among filters, so loading of heterologous filters in the same vial was avoided. The bonding reaction was performed at the desired temperatures.RNA filters were washed as follows: for single filters the condensing agent solution was removed by aspiration, replaced with a 1:1 mixture of formarmide-1.5 mM NaCl-0.15 mM Na citrate (pH 7) and incubated at 35d for 1 hr.The solution was removed by aspiration and washed with NaCl-Na citrate by filtration. For batch treatment, filters were incubated in the formamide solution as described above, then in NaCl-Na citrate by repeatedly removing the solution by aspiration and replacing it with fresh solution. Finally, all filters were washed with acetone and dried with N2.Either procedure removes 95% of the unreacted RNA.Determination of filter radioactivity was influenced by the nature of the filter paper, the amount of RNA on the filter, and the nature of the processing treatment. An attempt to correct for this differential quenching was made by spotting known aliquots of RNA on appropriate filters. 2975Abbreviation: COIm2, carbonyldiimidazole.

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A General Method for the Isolation of Rna Complementary to Dna

Cited by 266 publications

References 14 publications

The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-Dependent protein kinase

The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-Dependent protein kinase

FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis

Nucleic Acid Hybridization with RNA Immobilized on Filter Paper

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