Strand-specific Southern Blot
Strand-specific Southern Blot - Related Videos
02:57
Southern Blot
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2021
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments. Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
05:42
Southern Blotting to Detect Homologous Recombination in the Mouse Embryonic Stem Cell Genome
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2025
In this video, we describe the Southern blotting technique to identify homologous recombination events in the genome of mouse embryonic stem cells using radiolabeled probes. DNA regions containing homologous recombination events appear as distinct bands when the membrane containing the probe-bound DNA is exposed to X-rays.
09:40
Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
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Cited by 9 •
2011
The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.
08:51
Northern Blot to Detect and Quantify Specific MicroRNA in Total RNA Extract of Plant Tissue
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2025
In this video, we demonstrate the northern blot, a hybridization technique to detect and quantify microRNAs (miRNAs) from total RNA extracted from plant tissue.
09:04
Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids
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Cited by 12 •
2017
We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.
FAQs
Strand discrimination comes from the probe’s sequence and its hybridization behavior. Under controlled conditions, the labeled probe binds the complementary target strand in the DNA fragment, while the nonmatching strand does not produce the intended hybridization signal. Consequently, detection reports not only that a fragment contains the sequence, but also which strand orientation matches the probe.
Restriction digestion converts genomic or cloned DNA into defined fragments before separation. Each fragment can then be resolved by gel electrophoresis and transferred to a membrane, allowing the probe signal to be associated with a particular fragment rather than with an undifferentiated DNA sample. This organization helps connect strand-specific detection with DNA structure and arrangement.
Unlike a Southern blot interpreted only for sequence presence, a strand-specific Southern blot adds orientation information. A detected band indicates a restriction fragment that matches the probe, and the strand-specific design identifies which complementary strand carries the relevant sequence. That distinction is useful when sequence orientation or the arrangement of DNA within a construct matters.
A detected signal identifies the restriction fragment that hybridized with the labeled probe. Its position after gel separation links the signal to a fragment of the digested DNA, while the probe’s strand preference supplies orientation information. Interpreted together, fragment identity and strand matching can characterize DNA organization, rather than merely recording whether the sequence exists.
Both genomic DNA and cloned DNA are suitable targets, depending on the biological question. In genomic material, the assay can help examine organization or rearrangements. In cloned constructs, it can test sequence-specific integration and orientation. Thus, the same detection strategy can be applied to naturally occurring DNA and engineered or isolated DNA arrangements.
Changes in the pattern of probe-reactive restriction fragments can provide evidence about altered DNA organization, while strand-specific matching adds information about orientation. For sequence-specific integration, the assay helps determine whether the relevant sequence is present in a particular fragment and which complementary strand corresponds to the probe. These outcomes support molecular characterization of biological changes.