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Journal ArticleDOI

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

05 Nov 1975-Journal of Molecular Biology (J Mol Biol)-Vol. 98, Iss: 3, pp 503-517
TL;DR: This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
About: This article is published in Journal of Molecular Biology.The article was published on 1975-11-05. It has received 30291 citations till now. The article focuses on the topics: Restriction fragment & Gel electrophoresis.
Citations
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Journal ArticleDOI
TL;DR: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Abstract: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

53,030 citations

Journal ArticleDOI
TL;DR: A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described, and these "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

23,324 citations

01 Jan 1984
TL;DR: In this article, a technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described, where DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers.
Abstract: A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

21,435 citations

Journal ArticleDOI
TL;DR: Labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.

10,489 citations

Journal ArticleDOI
TL;DR: The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this electrophoretic transfer technique.

8,346 citations

References
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Journal ArticleDOI
Julius Marmur1
TL;DR: A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA, and Representative samples have been characterized for their thermal stability and sedimentation behaviour.

11,573 citations

Journal ArticleDOI
TL;DR: A simple method for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film, which is ten times more sensitive than conventional autoradiography of isotopes with higher emission energies.
Abstract: A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week. When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.

8,114 citations

Journal ArticleDOI
TL;DR: Extracts of Hemophilus influenzae strain Rd contain an endonuclease activity which produces a rapid decrease in the specific viscosity of a variety of foreign native DNA's; this enzyme was purified approximately 200-fold.

728 citations

Journal ArticleDOI
TL;DR: The five EcoRI† restriction sites in bacteriophage lambda DNA have been mapped and the DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in Agreement with their mobilities on polyacrylamide gels.

712 citations

Journal ArticleDOI
TL;DR: It is concluded that the method provides a satisfactory measurement of molecular weight, which is almost independent of the nucleotide composition of RNA at moderate salt concentrations.
Abstract: 1. The effects of changes in experimental conditions on the mobility of RNA in polyacrylamide-gel electrophoresis were investigated. 2. The linear relation between log(molecular weight) and electrophoretic mobility was shown to be independent within limits of salt or gel concentration. 3. The relative mobility of RNA with low content of guanylic acid and cytidylic acid residues was decreased in low-ionic-strength buffer. This was related to a small relative decrease in sedimentation coefficient. 4. However, Mg2+ ion caused almost no increase in mobility although it was associated with large increases in sedimentation coefficient. This suggested opposing actions of Mg2+ ion on the size and effective charge of the RNA. 5. It is concluded that the method provides a satisfactory measurement of molecular weight, which is almost independent of the nucleotide composition of RNA at moderate salt concentrations.

699 citations