| Principles of quenching by quencher |
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There are hydrolyzed probes and stem · loop structured probes among commonly used probes which make use of quenching by quencher. Principles of quenching by quencher is being explained in this column by citing the two as examples.
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Hydrolyzed Probe
While double-chain structure is formed after hybridization, fluorescent material is being quenched but once probes are decomposed by exonuclease activity of polymerase, quenching effect is cancelled and fluorescent light is emitted.
Stem · Loop Structured Probe
When probes having self-hairpin structure hybridize to the targets, fluorescent light is emitted when quenching effect is cancelled by elongation from the hair-pin structure.
That means "FRET quenching" of hydrolyzed probes would occur even if fluorescent material and quencher are about 40 base apart, but "Collisional quenching" of stem · loop structured probes would not occur unless the two are extremely adjacent each other.
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There are differences in quenching effect between hydrolyzed probes and stem · loop structured probes. The length of the both probes is 20 - 40mer and this difference does not come from difference of chain length.
If referring to the basic principles, quenching by hydrolyzed probe is called "FRET quenching" based on dipole · dipole structure and on the other hand,
quenching by stem · loop structure is called "Collisional quenching" based on electron exchange structure,
thus quenching is taking place in the two probes by different system.
That means "FRET quenching" of hydrolyzed probes would occur even if fluorescent material and quencher are about 40 base apart,
but "Collisional quenching" of stem · loop structured probes would not occur unless the two are extremely adjacent each other.
That means "FRET quenching" of hydrolyzed probes would occur even if fluorescent material and quencher are about 40 base apart,
but "Collisional quenching" of stem · loop structured probes would not occur unless the two are extremely adjacent each other.
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By the above mechanism, fluorescent light will not be emitted unless probes are discomposed in the case of hydrolyzed probes and in the case of stem · loop structure ,
it will switch on luminescent light by forming hairpin structure and it will switch off luminescent light by canceling hairpin structure.
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| < Principles of hydrolyzed probe > |
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| < Principle of Stem · Loop Structure Probe> |
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