Method of nucleic acid fragment detection

A method of nucleic acid fragment detection includes capturing a target nucleic acid fragment by an oligonucleotide probe to form a hybridised double strand. The oligonucleotide probe has an identification sequence complementary to the target nucleic acid fragment and a reproducible sequence. The hybridised double strand is removed to expose the reproducible sequence of the oligonucleotide probe. The repeats of the reproducible sequence are produced. The repeats of the reproducible sequence are labelled by a detection probe for identification and quantitation.

BACKGROUND

Field of Invention

The present invention relates to nucleic acid fragment detection method. More particularly, the present invention relates to a detection method of nucleic acid fragment detection with telomerase extension or polymerase replication.

Description of Related Art

MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules, consisting of 21-25 nucleotide bases. There have been many studies of miRNA regulation implicated in the etiology and progression of diseases, such as cancer, heart disease, and Parkinson disease. In addition, circulating miRNAs show great influence as a regulator in biological functions. The regulatory function of miRNAs affects cellular processes, such as proliferation or apoptosis, and correlation between miRNAs and cancer development is strong. Studies have shown that miRNAs are important biomarkers for different diseases. There is a strong need for a tool that can facilitate the analysis of the expression levels of the rapidly growing list of miRNA biomarkers that have been identified so far in the eukaryotic pool.

SUMMARY

In some embodiments, the instant disclosure provides a method of nucleic acid fragment detection. A target nucleic acid fragment is captured by an oligonucleotide probe to form a hybridised double strand. The oligonucleotide probe has an identification sequence complementary to the target nucleic acid fragment and a telomere sequence. The hybridised double strand is removed to expose the telomere sequence of the oligonucleotide probe. The repeats of the telomere sequence are produced. The repeats of the telomere sequence are labelled by detection probes.

In some embodiments, the oligonucleotide probe has an identification sequence complementary to the target nucleic acid fragment and a specific sequence that can serve as a primer for rolling circle amplification (RCA). The hybridised double strand is removed to expose the specific RCA primer sequence of the oligonucleotide probe. The repeats of the RCA sequence are produced via the extension of the said specific RCA primer sequence. The repeats of the RCA sequence are detected by detection probes. In some embodiments, the removal of the hybridised double strand includes cleaving the hybridized double strand off the oligonucleotide probe by duplex specific nuclease (DSN).

In some embodiments, the method includes identifying the target nucleic acid fragment according to a spatial resolution.

In some embodiments, the method includes providing a substrate having an immobile probe. The repeats of telomere sequence are captured through the immobile probe. The immobile probe has a sequence complementary to a segment of the oligonucleotide probe.

Due to the telomere repeats or RCA repeats, signal amplification is even more pronounced. Duplex-specific Nuclease (DSN) based enzymatic assay scheme identifies sequence of interest and exposes the reproducible sequence for extension and detection. This assay scheme is amenable to a fully automated process of nuclei acid fragment detection and quantitation. The target specific capture probes can be separately immobilized at different known locations on the substrate surface so as to obtain spatial resolution information. The signals from different locations on the substrate surface indicate the identity of different target nuclei acid fragments in the sample. In addition, the amount of target sequence can also be quantified.

The detection method can provide specific and sensitive multiplex detection of target fragment(s) from a variety of biological samples for use in biomedical research and clinical diagnostics applications.

DETAILED DESCRIPTION

Reference is made toFIG. 1, illustrating a flow chart of a method1000of nucleic acid fragment detection method in accordance with some embodiments of the instant disclosure. The method begins with operation1100in which a target nucleic acid fragment is captured by an oligonucleotide probe to form a hybridised double strand. The oligonucleotide probe has an identification sequence and a reproducible sequence. The identification sequence is complementary to the target nucleic acid fragment. The method continues with operation1200in which the hybridised double strand is removed to expose the reproducible sequence of the oligonucleotide probe. Subsequently, operation1300is performed. Repeats of the reproducible sequence are produced. The method continues with operation1400in which the repeats of the reproducible sequence are labelled by a detection probe. The method proceeds to operation1400in which the signals from the detection probe is analysed, and the target nucleic acid fragments are quantified according to the detected signals. The said detection probes can be labelled with various chemical or physical moieties that generate detectable signals under appropriate conditions. These detectable labels are well known in the art, comprising quantum dots, fluorescent dyes, or electrochemical molecules. The discussion that follows illustrates embodiments of nucleic acid fragment detection method according to the method1000ofFIG. 1. While method1000is illustrated and described below as a series of acts or events, it will be appreciated that the illustrated ordering of such acts or events are not to be interpreted in a limiting sense. For example, some acts may occur in different orders and/or concurrently with other acts or events apart from those illustrated and/or described herein. In addition, not all illustrated acts may be required to implement one or more aspects or embodiments of the description herein. Further, one or more of the acts depicted herein may be carried out in one or more separate acts and/or phases.

FIGS. 2 through 9illustrate various stages of a nucleic acid fragment detection method in accordance with some embodiments of the instant disclosure.

Reference is made toFIG. 2, illustrating a substrate having a plurality of oligonucleotide probes. The substrate210has a planar surface and bonded with a plurality of oligonucleotide probes200. Examples of materials of the substrate210include but not limited to, polystyrene, Polydimethylsiloxane (PDMS), glass, silicon or gold. In some embodiments, each of the oligonucleotide probes200is single strand and has an immobilization anchor, a reproducible sequence, and an identification sequence. Based on different identification sequences, the oligonucleotide probes200may have different types of oligonucleotide probes. For example, as shown inFIG. 2, the oligonucleotide probes200include first oligonucleotide probes202and second oligonucleotide probes204. Each of the first oligonucleotide probes202has the immobilization anchor212, reproducible sequence214, and a first identification sequence222. The immobilization anchor212allows the first oligonucleotide probes202to be attached on the substrate210surface through covalent bonds, for example. The 3′ end of the reproducible sequence214includes a telomerase recognition sequence. The first identification sequence222is attached to the 3′ end of the telomere sequence214. The first identification sequence222contains complementary sequence to a first type nucleic acid fragment and will attract the first type nucleic acid fragment binding.

Reference is still made toFIG. 2. Likewise, each of the second oligonucleotide probes204has the immobilization anchor212, the reproducible sequence214, and a second identification sequence224. The immobilization anchor212and the reproducible sequence214are identical to the first oligonucleotide probes202. In some embodiments, the reproducible sequence214is telomeres specific. That is, the reproducible sequence214allows telomerase recognition for amplification. The difference between the first oligonucleotide probes202and the second oligonucleotide probes204arises from the first identification sequence222and second identification sequence224. The second identification sequence224is attached to the 3′ end of the telomere sequence214, and the second identification sequence224contains complementary sequence to a second type nucleic acid fragment. Because of different identification sequences, the first oligonucleotide probes202and the second oligonucleotide probes204attract different types of nucleic acid fragments. The number of oligonucleotide probes may vary according to detection requirement, and the types of oligonucleotide probes may be one, two, three or more, and the instant disclosure is not limited thereto.

Reference is made toFIG. 3and operation1100ofFIG. 1, illustrating addition of a sample. The sample may include varied nucleic acid fragments. The nucleic acid fragments may be naturally occurring nucleotides and/or nucleotides that are not known to occur in nature. In some embodiments, the nucleic acid fragments are microRNA. In some embodiments, the sample includes first nucleic acid fragments322, second nucleic acid fragments326, and third nucleic acid fragments328as shown inFIG. 3. The first, second and third nucleic acid fragments322,326, and328may be the same or different type of nucleotides, and each type of the nucleic acid fragments has a sequence distinguishable from the other. That is, the first, second and third nucleic acid fragments322,326, and328are not identical. At least one or more bases are different therebetween.

Reference is made toFIG. 4and operation1100ofFIG. 1, illustrating formation of hybridised double strands. The first oligonucleotide probes202and second oligonucleotide probes204each contains a complementary sequence to the sequence of interest. In some embodiments, as shown inFIG. 4, the first nucleic acid fragments322contain sequence of interest. This sequence of interest makes the first nucleic acid fragments322the target nucleic acid fragment. The sequence of interest may reflect a high risk of certain disorders or a key cellular function, for example. It should be understood that the first nucleic acid fragments322may have a sequence length longer than the sequence of interest. The first identification sequences222of the first oligonucleotide probes202capture the first nucleic acid fragments322because the first identification sequences222are complementary to the sequence of interest of the first nucleic acid fragments322. While the second and third nucleic acid fragments326and328do not match the sequence of interest to either the first identification sequence222or the second identification sequence224. The second and third nucleic acid fragments326and328will not be retained (i.e., unbound) by the oligonucleotide probes200and are washed away.

Reference is still made toFIG. 4. The binding between the first identification sequence222and the first nucleic acid fragments322makes a portion of the single stranded first oligonucleotide probes202into a hybridised double strand. The first nucleic acid fragments322are captured by the first identification sequences222, and this combination results in hybridised double strands202a,202b, and202c. The remaining portion of the first oligonucleotide probes202, which includes the telomere sequence214and the immobilization anchor212, is still in single strand state as shown inFIG. 4. The second oligonucleotide probes204do not capture nucleic acid fragment that contains sequence of interest complementary to the second identification sequences224and remain single stranded on the substrate210surface.

Reference is made toFIG. 5and operation1200ofFIG. 1, illustrating addition of duplex-specific nuclease (DSN). The substrate210has double stranded and single stranded fragments. The first identification sequences222and the first nucleic acid fragments322are hybridised double strands202a,202b, and202c, while the second oligonucleotide probes204and the remaining portion of the first oligonucleotide probes202are single strands. The double-stranded state raises a flag to the duplex-specific nuclease402, while the single-stranded segments do not attract attention from the duplex-specific nuclease402.

Reference is made toFIG. 6and operation1200ofFIG. 1, illustrating the cleavage of the double strands. The duplex-specific nuclease402recognizes the hybridised double strands202a,202b, and202cand cuts the first identification sequences222and the first nucleic acid fragments322off from the first oligonucleotide probes202. The second oligonucleotide probes204are intact because they are single strands that do not initiate duplex-specific nuclease402into action. The entire second oligonucleotide probes204stand on the substrate210surface, while only portions of the first oligonucleotide probes202′ remain. More specifically, the hybridised double strands202a,202band202care cleaved, and the single stranded telomere sequences214and immobilization anchors212are left behind on the substrate210surface. The removal of the hybridised double strands202a,202band202calso results in exposure of the telomere sequences214of the first oligonucleotide probes202′. The exposure of the telomere sequences214is translated into positive of sequence of interest. The second identification sequences224remain tagging along the telomere sequences214of the second oligonucleotide probes204, such that the telomere sequences214of the second oligonucleotide probes204are not exposed. The exposure of the telomere sequence drives the subsequent reaction to take place.

Reference is made toFIG. 7and operation1300ofFIG. 1, illustrating extension of telomere sequence. The hybridised double strands202a,202b, and202care removed, and the telomere sequences214are exposed. Telomerase, which is also known as terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3′ end of telomere sequence. The telomere repeat sequence has 6 bases in human, for example. When telomerase412are added into the reaction vessel, the exposed telomere sequences214of the first oligonucleotide probes202′ provide the binding sites for the telomerase412. The telomere sequences214of the second oligonucleotide probes204are not exposed because the second identification sequences224are attached at the end. The telomerase412cannot find binding site on the second oligonucleotide probes204because the 3′ end of telomere sequences214of the second identification sequences224are occupied. This discrimination allows telomerase412to produce telomere repeats at the exposed 3′ end of the exposed telomere sequences214of the first oligonucleotide probes202′. The telomere repeats232extend from the 3′ end of the telomere sequences214of the first oligonucleotide probes202and may have thousands of the 6-base repeats.

In some embodiments, the presence of telomere repeats232indicates positive result of the existence of sequence of interest, while the second oligonucleotide probes204does not find complementary sequence of interest and remain negative (i.e., without the telomere repeat sequence). These positive and negative results are further visualized by the addition of detection probes422.

Reference is made toFIG. 8and operation1400ofFIG. 1, illustrating the addition and binding of the detection probes. The positive first oligonucleotide probes202′ are recognized by the detection probes422through the telomere or RAC repeats232and bind thereto. The binding of the detection probes to the said telomere or RAC repeats can be achieved by various means known in the art, comprising hybridization probes or ligation probes. The detection probes422may contain fluorescence signals that can be naked-eye visible under certain radiant wavelength. The detection probes422label the positive first oligonucleotide probes202′ and indicate the position of the cluster.

In some embodiments, the sequence of interest is visualized through rolling-circle amplification (RCA). Reference is made toFIG. 9and operation1300ofFIG. 1. Each of the probes300includes an immobilization anchor312and a primer sequence314. The difference between the probes200and probes300arises from the reproducible sequences214and the primer sequences314and316. Unlike the reproducible sequence214, the primer sequences314and316include a specifically designed primer sequence that can be extended through RCA. For example, after cleavage of the double strands, the primer sequences314are exposed as shown inFIG. 6and serves as a primer for further extension. As shown inFIG. 9, a circularized extension template342is then added to the sample and the RCA reaction is initiated. The added circularized extension template342includes a sequence that is complementary to the primer sequence314and has a unique sequence designed for target identification. The combination of the circularized extension template342and the primer sequence314results in repeats332on the substrate310and are identified by labelled detection probes as shown inFIG. 8. Again, the existence of repeats332of the circularized extension template342gives the positive indicator of the sequence of interest in the sample.

In some embodiments, the primer sequence314of the probe300may also contain a unique artificial nucleic acid sequence serving as an identification (ID) tag for the sequence of interest to be identified. Each of the ID tags is specific to the sequence of interest and can be identified in the subsequent detection steps. For example, as shown inFIG. 8, the primer sequences314and316contains different types of identification tags to their sequences of interest respectively. The method of identification and quantitation of the said ID tags are well known in the art, comprising probe hybridization, ligation and sequencing. The use of ID tags on the primer sequences314and316on the probes300enables simultaneous analysis of multiple sequences of interest.

Reference is made toFIG. 10, illustrating a perspective view of a substrate in accordance with some embodiments of the instant disclosure. The positive first oligonucleotide probes202′ are visualized by the detection probes422on the substrate210a, while the negative second oligonucleotide probes204cannot be seen on the substrate210abecause the negative second oligonucleotide probes204do not bind with the detection probes422. Different types of oligonucleotide probes are grouped together on the substrate210asuch that the positive result is accompanied with a spatial resolution. It should be understood that the immobilization anchors212and reproducible sequences214are identical to each of the oligonucleotide probes200. If there are four different types of oligonucleotide probes (i.e., four different sequences of interest), these four different types of oligonucleotide probes will be arranged in four clusters on the substrate210a. When the sequence of interest is present in a sample, the positive oligonucleotide probes will be visualized by the detection probes. By identifying the position of these positive oligonucleotide probes on the substrate210a, the sequence of interest can be determined.

Reference is made toFIG. 11, illustrating a perspective view of a substrate in accordance with some embodiments of the instant disclosure. The substrate210bis different from the substrate210adue to the cartridge design. In some embodiments, the substrate210bhas four cartridges112,114,116, and118. The cartridges112,114,116, and118are shallow depressions that are parallel to each other in a longitudinal direction on the substrate210b. Each of the cartridges112,114,116, and118contains a different type of oligonucleotide probe. The spatial resolution of the nucleic acid fragment detection is defined by the channels. For example, as shown inFIG. 11, the first oligonucleotide probes202are disposed in the cartridge112and the second oligonucleotide probes204are disposed in the cartridge114. In some embodiments, the second oligonucleotide probes204may be disposed in the cartridge116or118. After the positive oligonucleotide probes are visualized, the cartridge112shows the signal from the detection probes422. This information is translated into that the sequence of interest of the first oligonucleotide probes202is present in the sample, while the sequence of interest of the second oligonucleotide probes204are not. The spatial resolution arises from the lanes of cartridges112,114,116, and118.

In some embodiments, oligonucleotide probes300have different ID tags that are used for simultaneous analysis of multiple sequences of interest in the biological sample. Each of the ID tags is assigned as an identifier for a specific target sequence. In some embodiments, the oligonucleotide probes300having different ID tags do not be spatially isolated from each other on the substrate310. In some embodiments, the oligonucleotide probes300having different ID tags are spatially isolated from each other on the substrate310.

In some embodiments, the substrate210may be used as a quantitation tool for the target nucleic acid fragments in a sample. More specifically, the substrate210includes a plurality of wells and the total number of wells on the substrate is known by design. In addition each of the wells contains oligonucleotide probes200of the same sequence. A sample solution containing the target nucleic acid fragments of interest is randomly distributed among the wells on the substrate, with less than one copy of target nucleic acid fragment of interest in each well on average. Every positive well represents one strand of sequence of interest in the sample. The quantitation results can be obtained through Poisson statistical analysis of all positive and negative wells, similar to digital PCR analysis. This nucleic acid fragment detection method may be seen as a digital counting method of target of interest.

FIGS. 12 through 17illustrate various stages of a nucleic acid fragment detection method in accordance with some embodiments of the instant disclosure.

Reference is made toFIG. 12, illustrating a plurality of oligonucleotide probes600. The difference between the oligonucleotide probes600and the oligonucleotide probes200arises from the anchor portion. The oligonucleotide probes200have immobilization anchors212, while the oligonucleotide probes600are not fixed on a substrate. Each of the oligonucleotide probes600has an anchor612. The anchor612will not settle until a later stage in the nucleic acid fragment detection process. As a result, the oligonucleotide probes600float in the solution. The first oligonucleotide probes are designated as206, and the second oligonucleotide probes are designated as208. The oligonucleotide probes200and600have substantially the same structure, but the oligonucleotide probes600have anchors612instead of immobilization anchors212.

Reference is made toFIG. 12and operation1100ofFIG. 1, illustrating addition of a sample. In some embodiments, the sample includes first nucleic acid fragments322, second nucleic acid fragments326, and third nucleic acid fragments328as shown inFIG. 12.

Reference is made toFIG. 13and operation1100ofFIG. 1, illustrating formation of hybridised double strands. The first oligonucleotide probes206and second oligonucleotide probes208each contains a complementary sequence to the sequence of interest. In some embodiments, as shown inFIG. 13, the first nucleic acid fragments322contain sequence of interest. This sequence of interest makes the first nucleic acid fragments322the target nucleic acid fragment. The first identification sequences222of the first oligonucleotide probes206capture the first nucleic acid fragments322, while the second and third nucleic acid fragments326and328do not match the sequence of interest to either the first identification sequence222or the second identification sequence224. The second and third nucleic acid fragments326and328will not be retained by the oligonucleotide probes200and are washed away. The binding between the first identification sequence222and the first nucleic acid fragments322makes a portion of the single stranded first oligonucleotide probes206into a hybridised double strand.

Reference is made toFIG. 14and operation1200ofFIG. 1, illustrating addition of duplex-specific nuclease (DSN). The first identification sequences222and the first nucleic acid fragments322are hybridised double strands206a,206b, and206c, while the second oligonucleotide probes208and the remaining portion of the first oligonucleotide probes206are single strands. The double-stranded state raises a flag to the duplex-specific nuclease402, while the single-stranded segments do not attract attention from the duplex-specific nuclease402.

Reference is made toFIG. 14, illustrating the cleavage of the double strands. The duplex-specific nuclease402recognizes the hybridised double strands206a,206b, and206cand cuts the first identification sequences222and the first nucleic acid fragments322off from the first oligonucleotide probes206. The second oligonucleotide probes208are intact because they are single strands that do not initiate duplex-specific nuclease402into action. The removal of the hybridised double strands206a,206band206cresults in exposure of the 3′ end of telomere sequences214of the first oligonucleotide probes206′. The exposure of the 3′ end of telomere sequences214is translated into positive of sequence of interest. The exposure of the telomere sequence drives the subsequent reaction to take place.

Reference is made toFIG. 15, illustrating anchoring of oligonucleotide probes and addition of telomere sequence. A substrate710is provided. The substrate710has a plurality of anchor sites712. In some embodiments, different anchors on the substrate can be used for different sequences of interest in order to incorporate spatial separation for each sequence of interest. The anchor sites712contain complementary sequence to the anchors612so as to immobilize the floating oligonucleotide probes600. The oligonucleotide probes600recognize the anchor sites712through the anchors612and bind to the anchor sites712on the substrate710.

Reference is made toFIG. 16, illustrating extension of telomere sequence. After the hybridised double strands206a,206b, and206care removed, and the 3′ end of the telomere sequences214are exposed. When telomerase412are added into the reaction vessel, the exposed telomere sequences214of the first oligonucleotide probes206′ provide the binding sites for the telomerase412. The telomere sequences214of the second oligonucleotide probes208are not exposed because the second identification sequences224are attached at the end. The telomere repeats232extend from the 3′ end of the telomere sequences214of the first oligonucleotide probes206and may have thousands of the 6-base repeats but take no action to the second oligonucleotide probes208.

These positive and negative results are further visualized by the addition of detection probes422. Reference is made toFIG. 17, illustrating the addition and binding of the detection probes422. The positive first oligonucleotide probes206′ are recognized by the detection probes422through the telomere repeats232and bind thereto. The detection probes422may contain fluorescence signals that can be naked-eye visible under certain radiant wavelength. The detection probes422label the positive first oligonucleotide probes206′ and indicate the position of the cluster. The positive identification of telomere repeats232indicate positive result of the existence of sequence of interest, while the second oligonucleotide probes208does not find complementary sequence of interest and remain negative (i.e., without the telomere repeat sequence).

In summary, the disclosed invention provides a method for the identification and quantitation of miRNA fragments in a biological sample. The oligonucleotide probe uses identification sequence to capture sequence of interest and forms into double-stranded nucleic acid fragment. The double strand is then cleaved by duplex-specific nuclease and the 3′ end of the reproducible sequence of the oligonucleotide probe will be exposed. The exposure of the reproducible sequence indicates a positive result of the sequence of interest. Subsequently, the positive oligonucleotide probes are visualized by detection probes.