Rapid and accurate single nucleotide polymorphism detection by fluorophore-nucleic acid interactions

Abstract

The present disclosure relates generally to devices and methods for rapid and accurate diagnosis of single nucleotide polymorphisms. According to one or more embodiments, a system for identifying a single nucleotide polymorphism (SNP) comprises an oligonucleotide primer having a SNP recognition sequence, a fluorophore conjugated to the oligonucleotide primer at a conjugation site to form a fluorophore-conjugated oligonucleotide primer, a polymerase for extending the oligonucleotide primer to form a double-stranded deoxyribonucleic acid (DNA) amplicon upon binding to an oligonucleotide strand having a complementary sequence to the oligonucleotide primer, and wherein the fluorophore-conjugated oligonucleotide primer is the only primer in the system such that the fluorophore emits a fluorescent signal only when the fluorophore-conjugated oligonucleotide primer binds to a sequence specifically complementary to the SNP recognition sequence.

Description

Technical Field Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 623,527, filed January 22, 2024, which is incorporated herein by reference in its entirety.

[0002] The embodiments described herein generally relate to systems, apparatuses, and methods for the rapid and accurate diagnosis of single nucleotide polymorphisms (SNPs). More specifically, embodiments relate to systems, apparatuses, and methods including a deoxyribonucleic acid (DNA) extension and amplification platform with nucleic acid sequence recognition based on fluorophore-nucleic acid interactions. In other embodiments, this disclosure relates to systems, apparatuses, and methods for the detection of single nucleotide polymorphisms (SNPs) that include fluorophore-nucleic acid interactions and linear extension. Background Technology

[0003] The inherent complexity of existing diagnostic platforms presents numerous challenges in the field of molecular diagnostics, leading to confusion and inefficiency. Current routine techniques are associated with ambiguity, including issues such as false positives, false negatives, high costs, and challenges in data interpretation. For example, polymerase chain reaction (PCR) and next-generation sequencing (NGS) are two major platforms, each with its own inherent challenges. Overcoming these obstacles is crucial for advancing patient care and facilitating medical research in the areas of SNP identification and patient diagnosis and treatment.

[0004] The completion of the human genome sequence and the emergence of genomic SNP databases ushered in a transformative era in medicine, shifting from traditional methods to precision medicine. This modern paradigm relies on a growing understanding of SNPs and their relevance to a wide range of clinical phenotypes, including, but not limited to, infectious diseases, cancer, asthma, neuropsychiatric disorders, sickle cell disease, and drug metabolism conditions.

[0005] SNPs play a crucial role in disease prevention and treatment strategies. SNP variants involve subtle changes at the genetic level, typically single-base pair differences in the DNA sequence. These changes can be difficult to detect compared to the presence or absence of the entire pathogen.

[0006] A fundamental requirement for advancing precision medicine is the ability to rapidly and accurately detect SNPs and other gene mutations in patients. To support these requirements, the U.S. Food and Drug Administration (FDA) has mandated that the pharmaceutical industry publicly disclose SNP data evaluated during the drug licensing process.

[0007] Various SNP detection and analysis methods have been designed, each based on different principles. Traditional SNP detection methods often face challenges related to nonspecific amplification. Sanger sequencing, another mutation detection method, is also time-consuming and expensive, and most critically, limited by its limited sensitivity (the detection limit is approximately 20% of the frequency of mutated alleles). Common SNP detection methods are also frequently limited by sequence dependence, enzyme efficiency, or fidelity, and require highly trained technicians, sophisticated equipment, comprehensive sample preparation, and meticulous result interpretation.

[0008] This disclosure relates to systems, apparatus, and methods for accurately and effectively identifying or assessing the presence of SNPs in a given sample. Summary of the Invention

[0009] Systems and methods for identifying or evaluating genetic alterations, including single nucleotide polymorphisms (SNPs), are described. Importantly, systems and methods that enable accurate, specific, and rapid detection of SNPs in a cost-effective manner are described.

[0010] In some implementations, the system for detecting SNPs utilizes fluorophore-labeled specific primers. Fluorophore-labeled SNP-specific primers amplify the signal only when the primer extension precisely matches the target DNA sequence. This accuracy ensures the specificity and precision of detecting genetic variations. The system mitigates the problem of false positives by using SNP-specific primers for linear extension during loop-mediated amplification, instead of the conventional cycling method used in PCR. This method ensures that the signal originates solely from the mutant DNA itself, eliminating any influence from PCR artifacts. In some implementations, robust amplification can be achieved due to loop-mediated isothermal amplification (LAMP) cycling primers. With the aid of fluorophore-labeled SNP-specific LAMP loop primers, the system can achieve seamless linear extension in a single reaction.

[0011] According to one or more embodiments, a system for identifying single nucleotide polymorphisms (SNPs) includes: an oligonucleotide primer containing an SNP recognition sequence; a fluorophore conjugated to the oligonucleotide primer at a conjugation site to form a fluorophore-conjugated oligonucleotide primer; a polymerase for extending the oligonucleotide primer to form a double-stranded deoxyribonucleic acid (DNA) amplicon upon binding to an oligonucleotide chain having a complementary sequence to the oligonucleotide primer; and wherein the fluorophore-conjugated oligonucleotide primer is the only primer in the system, such that the fluorophore emits a fluorescent signal only when the fluorophore-conjugated oligonucleotide primer binds to a sequence specifically complementary to the SNP recognition sequence.

[0012] In some implementations, the system also includes an amplification platform.

[0013] In other embodiments, the amplification platform is a loop-mediated isothermal amplification (LAMP) platform.

[0014] However, in other embodiments, the fluorophore is in a quenched state when the oligonucleotide primer conjugated to the fluorophore does not extend.

[0015] In one or more embodiments, the fluorophore is quenched when the complementary strand formed in the double-stranded DNA amplicon contains a mismatch at the conjugation site.

[0016] In other embodiments, the fluorophore is quenched when the complementary strand formed in the double-stranded DNA amplicon contains mismatches in two or more bases at the conjugation site.

[0017] In some implementations, the fluorophore-conjugated oligonucleotide primers are circular primers.

[0018] However, in other embodiments, the fluorophore is conjugated with guanine (dG), adenine (dA), cytosine (dC), or thymine (dT).

[0019] In some embodiments, the fluorophore is conjugated to an oligonucleotide base, the oligonucleotide base being approximately 1 to approximately 5 nucleotides from the 3' end of the oligonucleotide primer.

[0020] In some implementations, the system does not include a reverse primer.

[0021] According to one or more embodiments, a method of treating a patient includes: obtaining a sample from the patient, said sample containing oligonucleotides; cleaving and preparing a sample for a assay to identify single nucleotide polymorphisms (SNPs); combining a fluorophore-conjugated oligonucleotide primer and a polymerase with the oligonucleotide, wherein the fluorophore is conjugated to the fluorophore-conjugated oligonucleotide primer at a conjugation site, and the fluorophore-conjugated oligonucleotide primer contains an SNP recognition sequence; determining whether the oligonucleotide in the sample contains an SNP based on whether the fluorophore emits a fluorescent signal, wherein the fluorophore-conjugated oligonucleotide primer is the only primer in said method, such that the fluorophore emits a fluorescent signal only when the fluorophore-conjugated oligonucleotide primer binds to a sequence specifically complementary to the SNP recognition sequence; and treating the patient if an SNP is detected in the sample.

[0022] In some implementations, determining whether oligonucleotides in a sample include fluorophores that remain quenched indicates a DNA double-strand mismatch.

[0023] In other embodiments, treating a patient includes molecular diagnosis of a condition or disease, wherein the condition or disease is, optionally, tuberculosis (TB), COVID-19, meningitis, encephalitis, congenital infection, sepsis, acute coronary syndrome, tissue compatibility problems, adverse drug reactions, preeclampsia, cancer, or any combination thereof.

[0024] However, in other embodiments, the method further includes employing a digital platform, wherein optionally, the digital platform is used for applications in circulating cell-free DNA, circulating cell-free RNA, circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), circulating pathogens, minimal residual disease (MRD), single-cell analysis, and / or spatial diagnostics.

[0025] According to one or more embodiments, a method for identifying single nucleotide polymorphisms (SNPs) in an oligonucleotide sample includes: combining an oligonucleotide primer conjugated to a fluorophore containing an SNP recognition sequence, a polymerase, and an oligonucleotide sample, wherein the fluorophore is conjugated to the fluorophore-conjugated oligonucleotide primer at a conjugation site; determining whether the oligonucleotide sample contains an SNP based on whether the fluorophore emits a fluorescent signal; and determining that the oligonucleotide sample contains an SNP when a fluorescent signal is emitted; wherein the fluorophore-conjugated oligonucleotide primer is the only primer in the method, such that the fluorophore emits a fluorescent signal only when the fluorophore-conjugated oligonucleotide primer binds to a sequence specifically complementary to the SNP recognition sequence.

[0026] In some implementations, the fluorophore-conjugated oligonucleotide primers are circular primers.

[0027] In other embodiments, the fluorophore-conjugated oligonucleotide primers also include one or more locked nucleic acids (LNAs).

[0028] In other embodiments, the method does not include the use of a reverse primer. Attached Figure Description

[0029] The following description is accompanied by the accompanying drawings. All descriptions are given by way of non-limiting examples that may be useful in understanding how to implement the described methods and compositions.

[0030] Figure 1A This is a diagram illustrating the design of SNP detection primers based on the implementation plan;

[0031] Figure 1B This is a diagram illustrating the detection principle used in a system for detecting gene alterations (SNPs), in which, according to the implementation scheme, the fluorophore becomes fluorescent when there is no mismatch between the primer and its annealing site on the sample DNA template.

[0032] Figure 1CThis is a diagram illustrating the detection principle used in a system for detecting gene alterations (SNPs), wherein, according to the implementation scheme, the fluorophore remains quenched when there is a mismatch between the primer and its annealing site on the sample DNA template;

[0033] Figure 2 This is a diagram of oligonucleotide primers conjugated to fluorophores during loop-mediated isothermal amplification (LAMP) according to the implementation scheme;

[0034] Figure 3 This is an illustration demonstrating the challenges of the traditional PCR process in SNP detection mechanisms;

[0035] Figure 4 This is a diagram depicting the amplification phase (initiation phase not shown) of a loop-mediated isothermal amplification (LAMP) process, demonstrating that SNP detection can be achieved using the LAMP process according to some implementation schemes; and

[0036] Figure 5 This is a diagram illustrating methods for identifying or evaluating genetic alterations such as SNPs in a sample according to certain implementation plans. Detailed Implementation

[0037] Certain exemplary embodiments will now be described to provide a comprehensive understanding of the principles of the structure, function, manufacture, and use of the devices and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the drawings are non-limiting exemplary embodiments, and that the scope of this disclosure is defined only by the claims. Features illustrated or described in connection with one exemplary embodiment may be combined with features of other embodiments. Such modifications and variations are intended to be included within the scope of this disclosure. Therefore, aspects and features of each embodiment may not be described for every embodiment, but such aspects and features apply to the various embodiments unless stated or understood to the contrary.

[0038] As used herein, the terms "patient" or "user" refer to any subject, including but not limited to plants, pathogens, animals (e.g., pets, farm animals, etc.), and humans. A patient may have a condition and / or disease, or be suspected of having a condition and / or disease, and is therefore receiving drug treatment. In some cases, the patient is a mammal, such as a human, premature infant, newborn, infant, adolescent, teenager, or adult. In some cases, as used herein, the term "patient" refers to a person (e.g., male, female, or child). In some cases, as used herein, the term "patient" refers to an experimental animal used in plant, pathogen, or animal model studies. A patient or subject may be of any age, sex, or combination thereof.

[0039] The term “treatment” refers to the application of a therapy in an effective (e.g., therapeutic) amount, manner, or pattern to improve a condition, symptom, ailment, or ailment-related parameters or the likelihood thereof.

[0040] As used in this article, the terms “essentially” or “basically” refer to a large or significant degree, but not a complete degree.

[0041] As used herein, the term “about” refers to any value, including integer and fractional parts, that varies within a maximum of plus or minus 10% of the value modified by the term “about”. In some embodiments, “about” refers to plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the value.

[0042] Current mainstream methods for detecting single nucleotide polymorphisms (SNPs), including hybridization-based methods and amplification arrest mutation systems (ARMS), have two significant limitations. Hybridization-based methods (including TaqMan probes and molecular beacons) utilize the principle of complementary base pairing to identify specific alleles. DNA probes with known sequences are designed to bind to complementary sequences in target DNA, and the strength of the hybridization depends on the degree of sequence matching, thus enabling SNP detection.

[0043] On the other hand, the ARMS method is a PCR-based approach that uses specially designed primers to selectively amplify DNA fragments containing specific SNP alleles, allowing differentiation of genotypes based on successful or failed amplification. Using allele-specific primers with a single nucleotide mismatch at the 3' end, the reduced DNA polymerase processing capacity due to the mismatch means these primers can only effectively amplify DNA fragments containing matching alleles. PCR products are visible on an agarose gel, where the presence or absence of an amplified band indicates the genotype of the SNP locus.

[0044] Due to the strong binding between guanine (G) and cytosine (C) bases, SNPs typically have minimal impact on hybridization in GC-rich regions. GC pairs form three hydrogen bonds, compared to two hydrogen bonds for adenine (A) and thymine (T) pairs. High GC content leads to the formation of stable secondary structures, such as hairpins and self-dimers, which hinder primer annealing and result in low amplification efficiency, making it difficult to reliably detect SNPs in these regions. Therefore, GC-rich regions are more stable and less susceptible to disruption caused by SNP variations. Consequently, identifying SNPs in GC-rich regions has proven quite challenging because SNPs have minimal impact on hybridization.

[0045] Therefore, one limitation of these SNP detection methods is that designing assays for GC-rich regions remains a significant challenge compared to AT-rich regions, as it is generally easier to design primers, probes, or DNA clips for the less stable AT-rich regions. Furthermore, enhanced specificity in AT-rich regions is possible, especially when enhanced specificity can be achieved through specific nucleotide modifications such as locked nucleic acids (LNAs) or peptide nucleic acids (PNAs). LNAs and PNAs are specific nucleotide modifications that significantly enhance the binding affinity of synthetic nucleic acid chains to their complementary DNA or RNA targets, primarily by altering the backbone structure to provide increased stability and specificity compared to native nucleic acids. LNAs achieve this by “locking” the sugar ring conformation with a bridge between the 2' and 4' carbons, while PNAs replace the phosphodiester backbone with a pseudopeptide backbone, giving it a neutral charge and thus improving binding affinity.

[0046] Another limitation of current strategies is their inherent fallibility, which causes them to rely on probability rather than absolute determinism. For example, in conventional hybridization-based methods, probes preferentially bind to mutant amplicones, but probes can also incorrectly bind to wild-type amplicones, producing false positive signals.

[0047] Similarly, in the ARMS method, mismatches in mutant-specific primers preferentially block the amplification of wild-type templates while allowing the amplification of mutant templates. However, the blocking of wild-type templates is never perfect, and therefore, false positive signals may occur when wild-type templates are amplified. Thus, regardless of the chosen mechanism, background wild-type alleles may be amplified, leading to false positive signals. The interpretation of results depends on carefully setting the cutoff value, which is susceptible to various factors, such as differences in PCR efficiency caused by variations in DNA quality and quantity between actual and control samples.

[0048] Therefore, faster and more accurate systems and methods are needed to diagnose genetic alterations, such as SNPs, to support timely medical decision-making and treatment.

[0049] The implementation schemes described herein overcome the aforementioned challenges and relate to primers, systems, and methods for the rapid and accurate identification, diagnosis, and treatment of SNP patients. The implementation schemes involve primers, systems, and methods comprising genetic and biochemical assays for detecting single nucleotide polymorphisms.

[0050] Some embodiments involve primers, systems, and methods incorporating an isothermal PCR platform that combines the speed of loop-mediated isothermal amplification (LAMP) with the precision of nucleic acid sequence recognition based on fluorophore-nucleic acid interactions. Other embodiments involve primers, systems, and methods for rapid, multiplex, portable, highly sensitive, and highly accurate detection of genetic alterations in relevant samples. Embodiments involve primers, systems, and methods for performing assays that provide fluorescence readings in a binary manner (“on” or “off”) and can be completed very rapidly, for example, in less than 30 minutes in some embodiments. In other embodiments, the assay can provide fluorescence readings in a binary manner and can be completed in less than 10 minutes.

[0051] In some embodiments, the primers, systems, and methods are implemented to identify low levels of SNPs in both DNA and RNA across various tissue types and quality species. For example, in some embodiments, the primers, systems, and methods are implemented such that a single mutation can still be identified when it is present in only 1% or more of the DNA or RNA. In some embodiments, the primers, systems, and methods can be adapted to impurities, enhancing versatility for field samples.

[0052] The implementation scheme relates to a sequence-specific detection system that includes a non-spontaneous interaction between a fluorophore and a nucleic acid. The system includes a mechanism for conjugating one or more fluorophores to nucleic acid bases or thymine (dT), guanine (dG), adenine (dA), or cytosine (dC) in a primer containing an SNP recognition sequence.

[0053] In one or more embodiments, the system and method include SNP detection primers having a fluorophore conjugated to thymine (dT), guanine (dG), adenine (dA), or cytosine (dC). In some embodiments, the system and method include SNP detection primers having a fluorophore conjugated to thymine (dT), guanine (dG), adenine (dA), or cytosine (dC) within about 1, about 2, about 3, about 4, or about 5 base pairs from the 3' end of the primer, the primer terminating at the 3' end with an internal quencher, cytosine (dC), or guanine (dG). According to some embodiments, the primer includes thymine (dT) located about 1, about 2, about 3, about 4, or about 5 base pairs from the 3' end of the oligonucleotide primer, the oligonucleotide primer being terminated at the 3' end with cytosine (dC) or guanine (dG), and the thymine (dT) being conjugated to a signal fluorophore.

[0054] Figure 1APrimer designs according to one or more embodiments are shown. In the first line, the desired primers are shown as oligonucleotides with an illustrative SNP detection sequence and a fluorophore (light bulb) conjugated to thymine (dT) located three bases from the 3' end. In this single-stranded primer structure, the fluorophore remains quenched due to photoinduced electron transfer caused by the adjacent 3' cytosine (dC). Although dC is shown, the 3' terminal base can also be guanine (dG). Each of guanine (dG) and cytosine (dC) is an electron donor that readily transfers electrons to the fluorophore upon photoexcitation, quenching the emission of the fluorophore.

[0055] Figure 1A The middle line shows a comparison of a primer that cannot be used as a suitable SNP detection primer as described in this paper. In this primer, the fluorophore is conjugated to a thymine (dT) position 11 bases from the 3' end, which logically is too far for intrinsic electron transfer and quenching of 3' cytosine (dC) to effectively quench the fluorophore via photoinduced electron transfer.

[0056] In the third line, another comparative example of primers that cannot be used as suitable SNP detection primers as described herein includes 3'-terminal thymine (dT). Although the fluorophore is conjugated to thymine (dT) immediately adjacent to the 3' end, as in the first line, where the fluorophore can be quenched by photoinduced electron transfer from an intrinsic electron-donating quencher, the 3'-terminal thymine (dT) is not an easily electron-donating quencher.

[0057] In some embodiments, the system further includes a mechanism for fluorophore excitation. Fluorophore excitation depends on a precise match between the complementary sequences of the oligonucleotides conjugated to the fluorophore and the annealing sites, used as primers; for example, dT pairs with adenine (dA), or dC pairs with dG. Through precise pairing and elongation by DNA polymerase, the fluorophore is excited and emits fluorescence at a longer wavelength, "turning on" the fluorescence signal. A detected fluorescence signal indicates a precise match. When the fluorophore remains quenched, the signal remains off, indicating a mismatch.

[0058] In some embodiments, the system and method further include a fluorescence detector that converts the emitted fluorescence signal into an electrical signal that is measured and analyzed. Fluorescence emitted from the fluorophore enables real-time monitoring of the amplification process, as the amount of amplified DNA increases with each amplification cycle. In some embodiments, the amount of amplified DNA is quantified by measuring the fluorescence signal in each cycle to determine a cycle threshold (Ct) inversely proportional to the DNA concentration. When the fluorophore is excited and activated, it emits light at a longer emission wavelength by absorbing light at the absorption wavelength, and this emission is detected by the fluorescence detector.

[0059] In one or more embodiments, the system further includes a mechanism for detecting mismatches between complementary sequences and primers (e.g., dT paired with dC or dG or dT). If the system detects a DNA double-strand mismatch (see...), Figure 1C Due to SNPs, the fluorophore remains quenched and the fluorescence signal is "off" within a specific range of the fluorophore conjugation site. In some embodiments, the specific range is within 2 bases. In other embodiments, the specific range is from about 1 to about 5 bases, including any number of bases contained therein. In other embodiments, the specific range may exceed 5 bases. In one or more embodiments, the specific range is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases, or any range between about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases.

[0060] Some embodiments involve primers having an oligonucleotide chain containing thymine (dT) within about 1 to 5 bases from a 3' end terminated with cytosine (dC) or guanine (dG). In some embodiments, a fluorophore is conjugated to the thymine (dT) of the oligonucleotide chain to form a fluorophore-conjugated oligonucleotide primer.

[0061] In one or more embodiments, the fluorophore-conjugated primers can function as loop primers in a LAMP platform.

[0062] In some embodiments, the oligonucleotide primer includes a fluorophore and an intrinsic electron-transfer base, namely a guanine (dG) or cytosine (dC) base located at the 3' end of the primer, and the fluorophore remains quenched until annealed with the complementary strand and extended by a polymerase with precisely matched base pairs. All electrons in the matched DNA duplex are fully paired, and there are no free electrons available to quench the conjugated fluorophore, which will emit fluorescence in the presence of a light source with an excitation wavelength. However, when mismatches exist in the vicinity of the conjugated fluorophore, unpaired electrons will continue to quench the fluorophore. Although cytosine (dC) and guanine (dG) bases may be present in the vicinity of the fluorophore in the extended DNA duplex, the intrinsic cytosine (dC) and guanine (dG) bases do not quench the fluorophore. Only the 3' terminal cytosine (dC) or guanine (dG) base can quench the fluorophore in the single-stranded oligonucleotide. In a double-stranded DNA double helix, regardless of the identification of the 3' base (whether it is cytosine (dC) or guanine (dG)), the fluorophore adjacent to the 5' end will be turned on, which illustrates the importance of the position of the fluorophore in the primer.

[0063] This document describes single-stranded SNP recognition primers in its implementation, which have a conjugated fluorophore within approximately five or fewer base pairs from the 3' end, contain guanine (dG) or cytosine (dC) at the 3' end, and meet the following two criteria. First, the primer remains quenched and is excited / becomes unquenched only when a double helix is ​​formed. The second criterion is that the fluorophore is not quenched / excited / fluoresces only when the fluorophore anneals to the template oligonucleotide and is elongated by DNA polymerase to form a double helix in which all base pairs precisely match the primer sequence. The fluorophore is located away from the 3' end under the following conditions. An unquenched / excited / fluorescent fluorophore signal indicates that the complementary oligonucleotide includes the SNP sequence or a perfectly matched DNA double helix. When a mismatch exists in the oligonucleotide at or within approximately five or fewer base pairs of the fluorophore, the fluorophore remains quenched, indicating that the oligonucleotide does not include the SNP sequence.

[0064] like Figure 1B and 1C As shown, in some embodiments, the system comprises a fluorophore conjugate capable of recognizing an SNP. The fluorophore conjugate is a fluorophoretically conjugated oligonucleotide primer. In these embodiments, the fluorophoretically conjugated oligonucleotide primer is terminated with dC or dG. In one or more embodiments, the primer includes a fluorophore modification on thymine (dT) adjacent to the 3' end of the primer. In other embodiments, the primer includes a fluorophore modification on dC, dG, dA, or dT.

[0065] When the fluorophore is attached closer to the 3' end of the DNA double helix, the dG / dC pair at the 3' end will quench the fluorophore. Therefore, an oligonucleotide containing a fluorophore is a primer that elongates to form a double helix, and the primer sequence (including the attached fluorophore) becomes the 5' end of the double helix (e.g., ...). Figure 1B and 1C (As shown). If the primers are only annealed to the complementary template but not extended, the fluorophore will remain close to the 3' end, and the dG / dC pair will continue to quench the fluorophore.

[0066] In some embodiments, the fluorophore-conjugated oligonucleotide primer comprises a fluorophore attached directly to the oligonucleotide base, or via a linker molecule. In one or more embodiments, the fluorophore is fluorescein. In other embodiments, the fluorophore-conjugated oligonucleotide primer comprises various fluorophores, including but not limited to fluorescein amidite, carboxy-X-rhodamine, hexachlorofluorescein, tetrachlorofluorescein, asymmetric sulfonate dyes (e.g., VIC fluorescein amidite, which fluoresces in the yellow-green portion of the spectrum), carboxy-tetramethylrhodamine (e.g., TAMRA), cyanine 3, cyanine 5, or any combination thereof.

[0067] In one or more embodiments, the fluorophore is directly attached to the oligonucleotide primer base. In other embodiments, the fluorophore is attached to the oligonucleotide primer base via a linker molecule.

[0068] In some embodiments, the fluorophore conjugation site is located approximately 2 to approximately 6 bases from the 3' end of the oligonucleotide primer. In other embodiments, the fluorophore conjugation site is located approximately 2, 3, 4, 5, and 6 bases from the 3' end of the oligonucleotide primer, or anywhere within such a range.

[0069] In one or more embodiments, the quenching group of the fluorophore-conjugated oligonucleotide primer is an electron donor. In some embodiments, the quenching group of the fluorophore-conjugated oligonucleotide primer is guanine (dG) or cytosine (dC).

[0070] In some embodiments, the fluorophore-conjugated oligonucleotide primer comprises about 10 to about 30 oligonucleotides. In other embodiments, the fluorophore-conjugated oligonucleotide primer comprises about 10 to about 100 oligonucleotides.

[0071] In one or more embodiments, the fluorophore-conjugated oligonucleotide primer further includes one or more locked nucleic acids (LNAs). In other embodiments, the fluorophore-conjugated oligonucleotide primer further includes one or more non-natural nucleic acids.

[0072] The fluorophore-conjugated oligonucleotide primers include an SNP recognition sequence that has a sequence that recognizes SNPs in an oligonucleotide sample. In one or more embodiments, SNP detection is achieved by using primers having a fluorophore conjugated to a nucleotide base, which utilizes specific base pairing to activate a fluorescence signal.

[0073] In some implementations, loop-mediated isothermal amplification (LAMP) is used to utilize fluorophore-nucleotide interaction mechanisms, as described below. Compared to conventional methods that lead to false positives, SNP-specific fluorophore-conjugated primers only participate in chain elongation but are not used as templates for subsequent amplification.

[0074] LAMP is a diagnostic DNA amplification technique that operates at an isothermal temperature (approximately 60–65°C), unlike PCR which requires thermal cycling. LAMP uses four to six primers to recognize six to eight distinct regions of the target DNA, performing a highly specific amplification reaction. Synthesis is initiated by strand displacement DNA polymerase, and two specially designed primers form a “loop” structure, which is facilitated by extension on the loop and additional annealing of the primers in subsequent rounds of amplification. The DNA product is a ladder-like band on an electrophoresis gel, formed by various repeats of short (e.g., 80–250 base pairs) target sequences linked to single-stranded circular regions in a long tandem.

[0075] In other implementations, other (non-LAMP) technology platforms may be used to leverage fluorophore nucleotide interactions of primers used for SNP detection.

[0076] In one or more embodiments, the system and method include an isothermal amplification platform.

[0077] In one or more embodiments, the system and method include an amplification platform in which the oligonucleotide primer is extended to form a double-stranded deoxyribonucleic acid (DNA) amplicon when the polymerase binds to an oligonucleotide chain having a sequence complementary to the oligonucleotide primer, wherein the fluorophore-conjugated oligonucleotide primer is the only primer in the system, such that the fluorophore emits a fluorescent signal only when the fluorophore-conjugated oligonucleotide primer binds to a sequence specifically complementary to the SNP recognition sequence.

[0078] like Figure 2 As shown, in some embodiments, the system and method include a LAMP process. Such a system and method includes fluorophore-modified SNP recognition primers. When the fluorophore-modified SNP recognition primer binds, combines, and anneals with a wild-type DNA strand that does not contain the corresponding SNP, the primer does not form a base pair match at the fluorophore-modified SNP site, forming a bump. The bump or DNA duplex mismatch between the SNP primer and the wild-type DNA strand causes the fluorophore to remain quenched or unexcited. No signal or fluorescence means that the sample or wild-type DNA strand does not contain the SNP. However, when a DNA strand containing a mutant SNP binds to and anneals with the SNP-modified recognition primer, and a complete pairing occurs between the primer and the DNA strand, the fluorophore is excited, turning on a signal or fluorescence. The detected fluorescence signal means that the sample or mutant DNA strand does indeed contain the SNP.

[0079] In some implementations, during loop-mediated isothermal amplification (LAMP), the fluorophore on the fluorophore-conjugated oligonucleotide primer is excited based on two key conditions. First, the fluorophore-conjugated oligonucleotide primer must undergo elongation or extension, resulting in the formation of a double-stranded DNA amplicon. Second, the complementary strand of the double-stranded DNA amplicon must precisely match the DNA strand carrying the fluorophore modification.

[0080] In some embodiments, when the fluorophore-conjugated oligonucleotide primer remains single-stranded and does not undergo elongation or extension, the conjugated fluorophore remains self-quenched or in a quenched state (and the fluorescence signal remains off). In other embodiments, when the fluorophore-conjugated oligonucleotide primer is elongated to form a double-stranded DNA amplicon, the conjugated fluorophore remains self-quenched, but the complementary strand contains a DNA double-strand mismatch at the fluorophore conjugation site. In other embodiments, when the fluorophore-conjugated oligonucleotide primer is elongated to form a double-stranded DNA amplicon, the conjugated fluorophore remains self-quenched, but the complementary strand contains a mismatch in the vicinity of the fluorophore conjugation site, for example, within about 1 to about 5 base pairs.

[0081] According to some implementation schemes, methods and systems for SNP detection do not include reverse primers. In other words, the fluorophore-conjugated primers only participate in chain elongation and are not used as templates for subsequent rounds of amplification. The fluorophore-conjugated oligonucleotide primers are the only primers in the system, so the fluorophore emits a fluorescent signal only when the oligonucleotide primers bind to a sequence that is specifically complementary to the SNP recognition sequence in the primer.

[0082] In one or more embodiments, the fluorophore-conjugated primers participate only in chain elongation and not in subsequent amplification (which can lead to false positives).

[0083] Contrary to the implementation described herein that requires specific base pair matching to turn the signal on and off, Figure 3 The results show that in conventional PCR, a signal is always generated regardless of the template used. This is because after the fluorophore-conjugated oligonucleotide primer binds to the template and is extended by polymerase, the resulting double-stranded DNA amplicons serve as templates for subsequent amplification. The reverse primer binds to the strand containing the fluorophore and uses it as a template to synthesize the complementary strand, ultimately producing a perfectly matched DNA duplex. At this point, the fluorophore becomes excitable because both of the aforementioned conditions are met. Therefore, conventional PCR systems can lead to false positives because the fluorescence signal always turns on after amplification.

[0084] In some embodiments of this disclosure, a multi-primer amplification system is used for SNP detection. Additional primers and competing primers may also be used to improve speed and / or eliminate false positives.

[0085] Figure 4This describes a multi-primer amplification system based on a LAMP system according to an embodiment. In some embodiments, multiple SNP-specific loop primers labeled with two or more fluorophores are used for amplification and linear extension. In one or more embodiments, robust amplification and seamless linear extension facilitated by fluorophore-labeled SNP-specific loop primers both occur in a single reaction.

[0086] The amplicons generated by the fluorophore-labeled SNP-specific loop primers used in the LAMP system are not used as templates for subsequent amplification. Cyclic amplification is achieved using forward inner primers (FIP), reverse inner primers (BIP), forward primers (F3), and / or reverse primers (B3). The fluorophore-labeled SNP-specific loop primers serve a dual purpose: accelerating the initiation phase and promoting the formation of dumbbell-shaped structures, which act as the starting point for robust cyclic amplification and extension. After this phase, the fluorophore-labeled SNP-specific loop primers can also bind to the loop region and be extended by the polymerase used in the LAMP system. However, the resulting amplicons are not used as templates, effectively avoiding the problems encountered in conventional PCR systems. Therefore, unlike conventional PCR systems, the multi-primer amplification system based on the LAMP system does not produce false positives. This method ensures that the signal originates only from the mutant DNA, i.e., DNA containing the SNP itself, excluding any influence from PCR artifacts. This method eliminates false positives by ensuring that non-specific amplification does not produce signals through linear extension using only one fluorophore-labeled SNP-specific loop primer during amplification.

[0087] In some embodiments, this disclosure relates to methods for identifying or evaluating SNPs in oligonucleotide samples. In some embodiments, such as Figure 5 As shown, the methods and systems include acquiring samples, lysing samples, preparing samples, and evaluating samples. In some embodiments, the samples include, but are not limited to, DNA, RNA, cell-free DNA (cfDNA), fresh tissue, frozen tissue, and formalin-fixed paraffin-embedded (FFPE) tissue.

[0088] In one or more embodiments, the systems and methods for identifying SNPs in a sample further include lysing the sample with a lysin and extending the primers with a polymerase.

[0089] In some implementations, the method for identifying or assessing SNPs also includes treating patients based on SNP testing. In one or more implementations, the treatment of a patient's condition or infectious disease includes, but is not limited to, tuberculosis (TB) (i.e., Mycobacterium tuberculosis (MTB)), COVID-19, the common cold, influenza (flu), COVID-19, gastroenteritis, hepatitis, respiratory syncytial virus (RSV), meningitis, encephalitis, congenital infection, sepsis, acute coronary syndrome, histocompatibility problems, adverse drug reactions, preeclampsia, or cancer, or any combination thereof.

[0090] In some embodiments, methods for identifying or evaluating SNPs are used as surgical applications for testing unspecified samples. In one or more embodiments, methods for identifying or evaluating SNPs are used as surgical applications for differentiating tumor types. In some embodiments, methods for identifying or evaluating both SNPs and structural variants are used as surgical applications for precise resection margin assessment. In other embodiments, methods for identifying or evaluating SNPs or structural variants are used as surgical applications for intraoperative confirmation of molecular characteristics. However, in some embodiments, methods for identifying or evaluating SNPs are used to conduct intraoperative clinical trials.

[0091] In one or more embodiments, the method for identifying or evaluating SNPs is used for the diagnosis and treatment of infections, including but not limited to upper respiratory tract infections, iatrogenic infections, vascular invasive infections, sexually transmitted infections, foodborne pathogen-related infections, or any combination thereof.

[0092] In some implementations, identification or SNP methods are used for liquid biopsy applications for cfDNA, including SNP, structural variants, copy number analysis, expression level profiling, methylation measurement, or any combination thereof.

[0093] In one or more embodiments, the method for identifying or evaluating SNPs and structural variants includes implementing any digital platform. In other embodiments, the digital platform is used for applications including, but not limited to, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), minimal residual disease (MRD), single-cell analysis, spatial diagnostics, or any combination thereof.

[0094] According to one or more embodiments, fluorophore-conjugated oligonucleotide primers are attached to the solid surface of a chip, and the sample hybridizes with the chip, allowing high-throughput detection of a set of mutations. Example

[0095] In standard intraoperative pathology, tissue biopsies are embedded in optimal cutting temperature compound (OCT) and subsequently evaluated pathologically on frozen sections and slides. To validate the technique described herein (hereinafter referred to as the SNPsnipe technique) with surgical samples, a retrospective preclinical relevance study was conducted at Duke University using OCT-embedded brain tissue preserved in liquid nitrogen at The Preston Robert Tisch Brain Tumor Center. A 10-micrometer-thick frozen section roll from each sample was lysed in 150 µl of SNPsnipe lysis buffer, and 4 µl of lysate supernatant was used for each SNPsnipe assay reaction. All tissue preparation and SNPsnipe assays were performed independently by the Duke Brain Tumor Biorepository and the Duke Molecular Physiology Institute.

[0096] Fifteen (15) patient brain tumor OCT samples with available NGS data were selected, including 5 glioblastomas (IDH1 wild-type, TERT C228T), 5 astrocytomas (IDH1 R132H, TERT wild-type), and 5 oligodendrogliomas (IDH1 R132H, TERT C228T). All samples were blinded using a SNPsnipe assay. The total run time for the SNPsnipe test was approximately 20 minutes, including 5 minutes for sample lysis and 15 minutes for reaction time. As shown in Table 1, both the IDH1R132H assay and the TERT C228T assay showed 100% (10 / 10) positive concordance (PPA) and 100% (5 / 5) negative concordance (NPA) relative to the NGS method. This result provides strong proof of principle for the accuracy and feasibility of using SNPsnipe assays on clinically relevant samples within the recommended timeframe (approximately 20 minutes from sample to result). Table 1. Comparison of SNP snip measurement and NGS sequencing results using clinical frozen samples

[0097] The accompanying drawings provided herein are not necessarily to scale; however, those skilled in the art will recognize that the drawings are to scale and / or that their usual dimensions are when the drawings are not to scale. While in some embodiments the movement of one component is described relative to another component, those skilled in the art will recognize that other movements are also possible. Furthermore, many terms may be used interchangeably throughout the disclosure, but this will be understood by those skilled in the art. Additionally, although features, aspects, or steps are described as “first” or “second,” this numerical order is generally arbitrary, and therefore such numbering is interchangeable. Furthermore, in this disclosure, components with similar numbers in various embodiments generally have similar characteristics when they have similar properties and / or are used for similar purposes. Finally, this disclosure includes illustrations and descriptions, including prototypes, benchtop models, or experimental designs. Those skilled in the art will recognize how to rely on and, in view of this disclosure, integrate the provided technologies, systems, devices, and methods into products.

[0098] While the concepts of this disclosure are susceptible to various modifications and alternatives, specific exemplary embodiments of this disclosure have been shown by way of example. However, it should be understood that this disclosure is not intended to limit the concepts to the specific forms disclosed; rather, it is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. Although this disclosure relates to specific embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the subject matter set forth in the appended claims.

Claims

1. A system for identifying single nucleotide polymorphisms (SNPs), comprising: An oligonucleotide primer containing an SNP recognition sequence; A fluorophore, which is conjugated to the oligonucleotide primer at a conjugation site to form a fluorophore-conjugated oligonucleotide primer; A polymerase for extending the oligonucleotide primer to form a double-stranded deoxyribonucleic acid (DNA) amplicon upon binding to an oligonucleotide chain having a complementary sequence to the oligonucleotide primer; and The oligonucleotide primer conjugated to the fluorophore is the only primer in the system, such that the fluorophore emits a fluorescent signal only when the oligonucleotide primer conjugated to the fluorophore binds to a sequence that is specifically complementary to the SNP recognition sequence.

2. The system according to claim 1, further comprising an amplification platform.

3. The system according to claim 2, wherein the amplification platform is a loop-mediated isothermal amplification (LAMP) platform.

4. The system of claim 1, wherein the fluorophore is in a quenched state when the oligonucleotide primer conjugated to the fluorophore does not extend.

5. The system of claim 1, wherein the fluorophore is in a quenched state when the complementary strand formed in the double-stranded DNA amplicon contains a mismatch at the conjugation site.

6. The system of claim 1, wherein the fluorophore is in a quenched state when the complementary strand formed in the double-stranded DNA amplicon contains mismatches in two or more bases at the conjugation site.

7. The system of claim 1, wherein the fluorophore-conjugated oligonucleotide primer is a circular primer.

8. The system of claim 1, wherein the fluorophore is conjugated with guanine (dG), adenine (dA), cytosine (dC), or thymine (dT).

9. The system of claim 8, wherein the fluorophore is conjugated to an oligonucleotide base, the oligonucleotide base being approximately 1 to approximately 5 nucleotides from the 3' end of the oligonucleotide primer.

10. The system of claim 1, wherein the system does not include a reverse primer.

11. A method of treating a patient, the method comprising: A sample was obtained from the patient, the sample containing oligonucleotides; The sample was cleaved and prepared for the determination of single nucleotide polymorphisms (SNPs); The oligonucleotide primers conjugated to the fluorophore and the polymerase are combined with the oligonucleotides, the fluorophore is conjugated to the oligonucleotide primers conjugated to the fluorophore at the conjugation site, and the oligonucleotide primers conjugated to the fluorophore contain an SNP recognition sequence. The method determines whether an oligonucleotide in a sample contains an SNP based on whether the fluorophore emits a fluorescent signal. The oligonucleotide primer conjugated to the fluorophore is the only primer in the method, such that the fluorophore emits a fluorescent signal only when the oligonucleotide primer conjugated to the fluorophore binds to a sequence that is specifically complementary to the SNP recognition sequence. as well as If an SNP is detected in the sample, the patient is treated.

12. The method of claim 11, wherein determining whether an oligonucleotide in the sample comprises determining whether the fluorophore remains quenched, indicating a DNA double-strand mismatch.

13. The method of claim 11, wherein treating the patient includes performing molecular diagnosis of the condition or disease, wherein the condition or disease is, optionally, tuberculosis (TB), COVID-19, meningitis, encephalitis, congenital infection, sepsis, acute coronary syndrome, tissue compatibility problems, adverse drug reactions, preeclampsia, cancer, or any combination thereof.

14. The method of claim 11, further comprising employing a digital platform, wherein optionally, the digital platform is used for applications in circulating cell-free DNA, circulating cell-free RNA, circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), circulating pathogens, minimal residual disease (MRD), single-cell analysis, and / or spatial diagnostics.

15. A method for identifying single nucleotide polymorphisms (SNPs) in an oligonucleotide sample, the method comprising: The oligonucleotide sample is combined with an oligonucleotide primer containing a fluorophore that recognizes the SNP sequence, a polymerase, and the oligonucleotide sample. The fluorophore is conjugated to the oligonucleotide primer at the conjugation site. Based on whether the fluorophore emits a fluorescent signal, it is determined whether the oligonucleotide sample contains SNPs; and When a fluorescent signal is emitted, it is determined that the oligonucleotide sample contains SNPs; The oligonucleotide primer conjugated to the fluorophore is the only primer in the method, so that the fluorophore emits a fluorescent signal only when the oligonucleotide primer conjugated to the fluorophore binds to a sequence that is specifically complementary to the SNP recognition sequence.

16. The method of claim 11, wherein the fluorophore-conjugated oligonucleotide primer is a circular primer.

17. The method of claim 11, wherein the fluorophore-conjugated oligonucleotide primer further comprises one or more locked nucleic acids (LNAs).

18. The method of claim 15, wherein the method does not include the use of a reverse primer.

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