Fluorescent Hydrolysis Probes
Fluorescent Hydrolysis Probes - Related Videos
04:10
Hydrolysis of an Ester - Concepts
0 Views •
2020
Triglycerides Triglycerides are triester molecules composed of a glycerol backbone that is bound to 3 fatty acids. These form the basis of many biological lipids that are found in vegetable oils and fats. Lipids that are isolated from plant material are the basis of vegetable oils, whereas lipids extracted from animals are used as lard or general sources of fats. A fatty acid molecule has a long carbon chain — between 12 and 20 carbons — with a weak organic acid at one end. Some fatty acids...
11:04
Hydrolysis of an Ester - Student Protocol
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2020
Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA Preparation of SoapExpand Soap is prepared using a saponification reaction, where a base catalyzes the hydrolysis of three ester groups of an oil, such as coconut oil. During saponification, hydroxide ions from the base attack the carbonyl group on the oil to form a ratio of three molecules of soap to one molecule of glycerol. The resulting soap molecule is a long carbon chain, which is...
01:08
Hydrolysis of ATP
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2019
The bonds of adenosine triphosphate (ATP) can be broken through the addition of water, releasing one or two phosphate groups in an exergonic process called hydrolysis. This reaction liberates the energy in the bonds for use in the cell—for instance, to synthesize proteins from amino acids. If one phosphate group is removed, a molecule of ADP—adenosine diphosphate—remains, along with inorganic phosphate. ADP can be further hydrolyzed to AMP—adenosine monophosphate—by the removal of a second...
01:22
Hydrolysis
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2026
Hydrolysis is a chemical reaction in which the addition of water breaks down a polymer into its simpler monomer units. For example, peptides break into amino acids, carbohydrates into simple sugars, and DNA into nucleotides. Enzymes often facilitate these processes.Hydrolysis Reverses Dehydration SynthesisComplex carbohydrates can be broken down by breaking the bonds between individual sugar units. The reaction breaks a glycosidic bond as water is added to the compound. The glycosidic bonds...
04:02
An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells
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2025
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.
FAQs
The key event is separation of the reporter fluorophore from the quencher during amplification. While the probe remains intact, their proximity suppresses reporter emission. After the probe hybridizes to its complementary target, DNA polymerase uses 5′ nuclease activity to cleave it, disrupting that arrangement and allowing fluorescence to accumulate. This links enzymatic processing to target detection.
Probe design determines which amplified sequence produces a signal. A probe that matches the intended target can support selective detection, while sequence differences can prevent equivalent hybridization and signal generation. This selectivity is especially useful when assays must distinguish closely related sequences, including variants or mutation-containing targets, rather than measuring amplification from any related nucleic acid.
During quantitative PCR, fluorescence rises as cleaved probes release reporter emission. The accumulating signal reflects the amount of amplified target, so measurements can be used to quantify nucleic acid rather than simply record whether amplification occurred. Interpretation therefore depends on following signal development during the amplification process and relating it to the target being measured.
An assay brings together the target nucleic acid, amplification reaction, sequence-specific probe, fluorescent reporter, quencher, and DNA polymerase with 5′ nuclease activity. During the run, fluorescence is monitored as amplification proceeds. Coordinating these components allows the experiment to connect target-specific hybridization and probe cleavage with a measurable quantitative signal.
Researchers can apply these probes to gene expression analysis and pathogen detection, where measured fluorescence indicates accumulation of the corresponding amplified target. Their sequence selectivity makes them useful when the objective is not only to detect nucleic acid, but also to assess a particular biological target within an amplification-based assay.
For mutation or variant analysis, the probe sequence can be chosen to discriminate closely related nucleic acid sequences. A matching target produces the intended cleavage-associated fluorescence pattern, whereas sequence differences can alter whether the probe supports specific detection. This makes the approach useful for identifying variants and validating biological assays that depend on target identity.