Topic
Southern blot
About: Southern blot is a research topic. Over the lifetime, 9303 publications have been published within this topic receiving 389093 citations. The topic is also known as: Southern blotting.
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TL;DR: Results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.
3,994 citations
TL;DR: Three distinct human endothelin-related genes were cloned by screening a genomic DNA library under a low hybridization stringency with a synthetic oligonucleotide probe encoding a portion of the endotheli sequence, suggesting the possible existence of endothelins receptor subtypes.
Abstract: Three distinct human endothelin-related genes were cloned by screening a genomic DNA library under a low hybridization stringency with a synthetic oligonucleotide probe encoding a portion of the endothelin sequence. Genomic Southern blot analysis with the same oligonucleotide probe showed three corresponding chromosomal loci not only in the human genome but also in porcine and rat genomes. The nucleotide sequences of the three human genes were highly conserved within the regions encoding the 21-residue (mature) endothelins, in spite of the fact that the immediately upstream exon sequences, which encode a part of the propeptides, retained little similarity. Moreover, each of the human genes predicted a putative 21-residue peptide, similar to but distinct from each other: (i) the "classical" endothelin (ET-1), (ii) [Trp6,Leu7]endothelin (ET-2), and (iii) [Thr2,Phe4,Thr5,Tyr6, Lys7,Tyr14]endothelin (ET-3). Synthetic ET-1, ET-2, and ET-3 were prepared according to the deduced amino acid sequences, and the biological activities were assayed by contraction of isolated porcine coronary artery strips and by intravenous injection to anesthetized rats. All these synthetic peptides produced strong vasoconstrictor and pressor responses. However, the quantitative profiles of the pharmacological activities were considerably different among the three isopeptides, suggesting the possible existence of endothelin receptor subtypes.
2,548 citations
TL;DR: To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform.
2,483 citations
TL;DR: Polymerase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts.
Abstract: CYTOGENETIC abnormalities of chromosome 9p21 are characteristic of malignant melanomas1,2, gliomas3, lung cancers4 and leukaemias5. From a panel of 46 human malignant cell lines, we localized by positional cloning the most frequently deleted region on 9p21. Sequence analysis of the isolated fragment reveals two open reading frames identical to the recently described complementary DNA for the inhibitor of cyclin-dependent kinase 4 (CDK4) 6. Polymerase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts. One carcinoma had a deletion entirely within the CDK4-inhibitor gene. The CDK4-inhibitor gene from a patient with dysplastic nevus syndrome had a germ-line nonsense mutation. The CDK4 inhibitor is thought to be a physiological suppressor of proliferation. Cells unable to produce the inhibitor may be prone to neoplastic transformation.
1,829 citations
TL;DR: It is found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb.
Abstract: The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.
1,667 citations