Nucleotide Detection via Cyclic Exonuclease Signal Amplification
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Solution Overview
Problem
Current methods for sequencing DNA or RNA and detecting methylated and un-methylated nucleotide bases are inefficient due to weak fluorescence signals from nucleotide-specific reactive labels or incorporation into fluorescence-enhancing matrices, making reliable identification challenging.
Innovation Solution
A method involving progressive pyrophosphorolysis to generate a stream of single nucleotides, which are then reacted with a probe system comprising labeled oligonucleotides to form a double-stranded probe. This probe is treated with restriction endonucleases to differentiate modified and unmodified nucleotides, with exonucleolytic digestion releasing detectable elements for enhanced fluorescence signal detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleotide-specific reactive labels are used for sequencing, then nucleotide identification is enabled, but the fluorescence signal is too weak for reliable detection
Solution Approach 1:
The patent employs cyclic exonucleolytic digestion where the probe undergoes repeated cycles of exonuclease treatment and regeneration. Each cycle releases additional fluorophores, creating a periodic amplification effect that accumulates signal intensity over multiple cycles, transforming the initially weak single-event signal into a strong cumulative signal suitable for reliable detection
Solution Approach 2:
The patent changes the temporal parameter of signal detection by extending the measurement period through multiple cyclic digestion steps. Instead of detecting a single weak signal event, the system accumulates signal over time through repeated cycles, effectively increasing the total detectable fluorescence intensity while maintaining nucleotide identification precision
2Measurement precision
If nucleotides are incorporated into fluorescence-enhancing matrices, then detection is enabled, but the signal remains too weak for reliable identification
Solution Approach 1:
The cyclic exonucleolytic digestion process repeatedly exposes the probe to exonuclease treatment, with each cycle releasing additional fluorophores from the probe structure. This periodic action accumulates fluorescence signal over multiple cycles, transforming the insufficient single-cycle signal into a robust cumulative signal that enables reliable nucleotide identification
Solution Approach 2:
The patent maintains continuous useful action by regenerating the probe after each digestion cycle and immediately subjecting it to another cycle of exonucleolytic digestion. This continuous cyclic process ensures that the fluorescence signal generation never stops, continuously accumulating detectable signal intensity throughout the detection process
3Productivity
If progressive pyrophosphorolysis is used to generate single nucleotides, then sequencing capability is improved, but the method complexity increases
Solution Approach 1:
The probe design incorporates multiple functional elements: nucleotide capture capability, fluorophore carrying capacity, and exonuclease susceptibility. The cyclic exonucleolytic digestion process serves multiple purposes simultaneously: it releases fluorophores for detection, regenerates the probe for another cycle, and provides signal amplification. This multi-functionality reduces the need for separate systems for each function, managing complexity while enhancing sequencing capability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for reliable and efficient detection of nucleotide modifications, such as methylated cytosine or adenine, by amplifying the fluorescence signal through cyclic exonucleolytic digestion, enabling precise sequencing and methylation site localization.
Implementation Method 1
In each of such probe types, one of the two oligonucleotide components comprises characteristic fluorophores and in the probe's unused state the ability of these fluorophores to fluoresce remains extinguished by virtue of the presence of quenchers located close-by or by self-quenching
Implementation Method 2
digesting the first oligonucleotide strand of the used probe with an enzyme having double-stranded exonucleolytic activity in the 3'-5' direction
Implementation Method 3
treating the used probe with modification-dependent restriction endonuclease to cut only the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is modified
Implementation Method 4
reacted, in the presence of a polymerase and a ligase, one of the single nucleotides with a corresponding probe system
Implementation Method 5
reacted, in the presence of a polymerase and a ligase, one of the single nucleotides with a corresponding probe system
Implementation Method 6
generating a stream of single nucleotides by progressive pyrophosphorolysis of the nucleic acid
Data Source
Figure 1~2
AI summary
A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleotides by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, one of the single nucleotides with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with first and second regions of characteristic detectable element types in an undetectable state located respectively on the X' and Y' end sides of a third region comprising a restriction enzyme recognition site element including the capture site and an exonuclease-blocking site on the X' side thereof (wherein either X' is 3' and Y' is 5' or X' is 5' and Y' is 3') and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide flanking the capture site; (2a) either (i) treating the used probe with a conventional or nicking substitution-dependent restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is substituted or (ii) treating the used probe with a conventional or nicking substitution-sensitive restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is unsubstituted; (3) digesting the first oligonucleotide strand of the used probe with an enzyme having double-stranded exonucleolytic activity in the X'-Y' direction corresponding to the first oligonucleotide to yield detectable elements derived from either the first region, the second region, or the first and second regions in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating steps (2a), (3) and (4) in a cycle and (6) detecting the detectable elements released in each iteration of step (3) wherein if the endonuclease employed is of the conventional type the second or third oligonucleotide includes an endonucleolysis directing linkage at or close to its X' or Y' end respectively. Corresponding biological probe systems are also disclosed.