Patent Application
20240279728
The present disclosure relates generally to a detecting a dinucleotide sequence in a target polynucleotide.
Sequencing technologies, such as Sanger or Next-Generation Sequencing, are the most common methods to detect genomic sequences and variants of interest. Despite their high accuracy, these technologies remain time-consuming and expensive. There is currently no rapid and cost-efficient method that can be efficiently conducted using all-in-one reactions to detect desired genetic signatures.
Identifying variations in DNA sequences is a routine task in basic research for genetic testing, clinical diagnostic, or forensic purposes. Over the past decades, several technologies utilizing Sanger or Next-Generation Sequencing (NGS) platforms have been developed to facilitate the sequencing of DNA molecules, enabling the determination of DNA sequences and the identification of variants. However, although these technologies are easily accessible, as companies offer genomic platforms and sample processing, they remain time-consuming (several days to weeks) and expensive (from several dollars to thousand dollars) for routine laboratory experiments. Moreover, it requires the processing of samples by third parties, which can cause errors and contaminations during sample manipulation. The past two decades have also witnessed the development of an accelerated number of new techniques using variant-specific primers or probes. However, these techniques are not robust because the efficacy and specificity of the detection are strongly dependent on the sequence and mutation. In addition, these approaches lack specificities as they rely on weak and transient nucleic acid interactions to distinguish between genetic variants, which often differ from the reference by only one nucleotide.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the coronavirus disease 2019 (COVID-19), has spread rapidly becoming a global pandemic1-3. Despite the implementation of government-imposed mitigation measures, nationwide lockdowns, and worldwide travel bans, COVID-19 has caused more than 3 million deaths and is responsible for a high incidence of long-lasting COVID-19 symptoms4-6. Surveillance of circulating viruses revealed the emergence of variants carrying multiple concerning mutations7,8 capable of partially evading immune response, enhance virus transmission, and disease severity9-22.
The detection of SARS-CoV-2 nucleic acids in patient samples employs RT-PCR. However, RT-PCR does not identify variants23.
Genomic surveillance strategies for SARS-CoV-2 variants are primarily limited to the sequencing of viral nucleic acids isolated from infected humans24,25. Rapid detection methods to detect the presence of variants utilize mutation-specific primers and probes26-28. However, these approaches inherently exhibit low specificity because they rely on weak nucleic acid interactions to discriminate variants with only a single nucleotide difference to the reference.
The sequencing of SARS-CoV-2 genomes plays a fundamental role in the discovery of new emerging variants24,25. However, sequencing cannot substitute for the development of rapid routine tests for circulating variants. Indeed, sequencing requires sophisticated technologies29,30, has a high error rate requiring the deployment of complex bioinformatic pipelines31, is expensive, slow (several days), and susceptible to contaminations32. Therefore, the implementation of reliable, rapid, and cost-effective diagnostic tools into standard diagnostic platforms is needed to contain the propagation of the variants.
Despite the importance of detecting genetic sequences and associated mutations, there are still no all-in-one assays available for rapid detection of genetic signatures for routine laboratory experiments.
As described herein, there is provided:
In one aspect there is provided a method of detecting a mutation in a target sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotide sample, the method comprising:
In one example, wherein subjecting the Acu1-tagged amplicon to a digestion with Acu1 to generate a digestion reaction mixture comprising a digested Acu1-tagged amplicon is for about 1 minute at about 37° C.
In one example, wherein said heat inactivation steps comprises heating for about 1 minute to about 10 minutes at about 65° C.
In one example, wherein step (d) comprises a first step for about 10 minutes at about 65° C., a heating step for about 10 minutes.
In one example, the conditions to ligate said one or more adaptors comprises using T4 ligase or T3 ligase
In one aspect there is provided a method of detecting a mutation in a target sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotide sample, the method comprising:
In one example, wherein the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, the heat inactivation step comprises heating for about 1 minute at about 65 C.
In one example, wherein the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, wherein the heat inactivation step comprises heating for about 1 minute at about 65° C.
In one example, wherein the at least one primer polynucleotide further comprises a quencher and said one or more variant adaptors comprise a fluorophore.
In one aspect there is provide a kit comprising an adaptor, a container, and optionally instructions for the use thereof, said adaptor comprising a double-stranded DNA formed by the annealing of two complementary oligonucleotide; one of the two strand contains a 3′ dinucleotide overhang that is used to capture the complementary variant signature.
In one aspect there is provided a kit comprising one or more isolated polynucleotide selected from:
In one aspect there is provided a method for detecting a mutation in a target sequence of an infectious agent polynucleotide sample, the method comprising:
In one example, wherein the infectious agent is Deltavirus, Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Peribunyaviridae, Phenuviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Smacoviridae, or Togaviridae
In one example, wherein subjecting the Acu1-tagged amplicon to a digestion with Acu1 to generate a digestion reaction mixture comprising a digested Acu1-tagged amplicon is for about 1 minute at about 37° C.
In one example, wherein said heat inactivation steps comprises heating for about 1 minute to about 10 minutes at about 65° C.
In one example, wherein step (d) comprises a first step for about 10 minutes at about 65° C., a heating step for about 10 minutes.
In one example, wherein the conditions to ligate said one or more adaptors comprises using T4 ligase or T3 ligase
In one aspect there is provided a method for detecting a mutation in a target sequence of an infectious agent polynucleotide sample, the method comprising:
In one example, wherein the infectious agent is Deltavirus, Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Peribunyaviridae, Phenuviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Smacoviridae, or Togaviridae
In one example, wherein the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, wherein the heat inactivation step comprises heating for about 1 minute at about 65 C.
In one aspect there is provided a method for detecting a mutation in a target sequence of an infectious agent polynucleotide sample, the method comprising:
In one example, wherein the infectious agent is Deltavirus, Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Peribunyaviridae, Phenuviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Smacoviridae, or Togaviridae
In one example, wherein the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, wherein the heat inactivation step comprises heating for about 1 minute at about 65° C.
In one example, wherein the at least one primer polynucleotide further comprises a quencher and said one or more variant adaptors comprise a fluorophore.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Coronaviruses are a large family of viruses which cause illness in animals or humans. In humans, several coronaviruses are known to cause respiratory infections ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
Most recently identified is the 2019 novel coronavirus (SARS-CoV-2 (SCoV2)/COVID-19).
Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, was characterized as a pandemic by the World Health Organization (WHO) in March 2020 and has triggered an international public health emergency
Globally, as of 6 Jun. 2021, there have been 172,630,637 confirmed cases of COVID-19, including 3,718,683 deaths, reported to WHO.
A number of variants of SARS-CoV-2 have been identified.
Variants are viruses that have changed or mutated. Variants are common with coronaviruses. A variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral.
Mutations refers to nucleotide or amino acid substitutions, insertions or deletions, from the wild type (also referred to as reference) sequence. The term mutant or variants may encompass natural biological variants (e.g. allelic variants or geographical variations).
Thus, as used herein, the terms “variant polynucleotide” and “mutated polynucleotide” refer to one or more changes of a nucleic acid sequence of DNA or RNA, including, but not limited to a base substitution, insertion, deletion, reverse position, overlap, or the like
A SARS-CoV-2 isolate is a Variant of Interest (VOI) if, compared to a reference isolate, its genome has mutations with established or suspected phenotypic implications, and either: has been identified to cause community transmission/multiple COVID-19 cases/clusters, or has been detected in multiple countries; or is otherwise assessed to be a VOI by (for example) WHO in consultation with the WHO SARS-CoV-2 Virus Evolution Working Group.
As of 6 Jun. 2021, variants of interest include the following.
A SARS-CoV-2 variant of concern (VOC) is a variant that meets the definition of a VOI and, through a comparative assessment, has been demonstrated to be associated with one or more of the following changes at a degree of global public health significance: Increase in transmissibility or detrimental change in COVID-19 epidemiology; or Increase in virulence or change in clinical disease presentation; or Decrease in effectiveness of public health and social measures or available diagnostics, vaccines, therapeutics.
As of 6 Jun. 2021, variants concern include the following.
Other naming systems are being developed for variants of SARS-CoV-2.
In one example, there is provided a method of detecting a mutation in a target sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotide sample, the method comprising:
In one example, subjecting the Acu1-tagged amplicon to a digestion with Acu1 to generate a digestion reaction mixture comprising a digested Acu1-tagged amplicon is for about 1 minute at about 37° C.
In one example, said heat inactivation steps comprises heating for about 1 minute at about 65° C.
In one example, wherein step (d) comprises a first step for about 10 minutes at about 25° C., a heating step for about 10 minutes.
In one example, the conditions to ligate said one or more adaptors comprises using T4 ligase or T3 ligase
In one aspect there is provided a method of detecting a mutation in a target sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotide sample, the method comprising:
In one example, the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, the heat inactivation step comprises heating for about 1 minute at about 65° C.
In one aspect there is provided a method of detecting a severe acute respiratory syndrome coronavirus in a sample, the method comprising:
In one example, wherein the reaction conditions of step (b) are carried out for about 10 minutes at room temperature.
In one example, the heat inactivation step comprises heating for about 1 minute at about 65° C.
In one example, wherein the at least one primer polynucleotide further comprises a quencher and said one or more variant adaptors comprise a fluorophore.
In another aspect, a Type IIS restriction enzyme-tagging primer polynucleotide is used. Specific examples of Type IIS restrictions enzymes include Acu1, Bpml, BpuEl, Bsgl, Mmel, and NMeAlll. In one aspect, there is provided a method for detecting a mutation in a target sequence of an infectious agent polynucleotide sample.
In some examples, the infectious agent is Deltavirus, Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Nairoviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Peribunyaviridae, Phenuviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Smacoviridae, or Togaviridae
The term “detect” or “detecting” refers to identifying the presence, absence, or amount of the nucleic acid to be detected.
The term “mutation”, as used herein, refers to any change in a nucleic acid fragment relative to the “normal” (or wild type or reference) genetic material. The nucleotide sequence of the mutated nucleic acid herein displays one or more differences from the nucleotide sequence of the corresponding, non-mutated nucleic acid. A mutation may be one or more of a deletion, insertion, or substitution of one or more nucleotides.
The term “variants”, as used herein, includes nucleic acids and proteins whose sequence varies from the sequence of a reference nucleic acid and protein
Thus, in some examples a mutant may also be referred to as a variant.
The term “target sequence”, as used herein, refers to the region of interest on the original DNA. In some examples, the target sequence comprises the location(s) of the sequences of a VOI or VOC.
Accordingly, in some aspects, there is described herein a method of detecting mutants or variants of SARS-CoV-2.
The term “polynucleotide”, as used herein, refers to a single or double stranded polymer composed of nucleotide monomers.
The term “nucleic acid”, as used herein, refers a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.
The terms “ribonucleic acid” and “RNA”, as used herein, refers to a polymer composed of ribonucleotides.
Reference to the expression “5” end of a sequence segment refers to the localization of the sequence of nucleotides referred to is towards the 5′ terminal end of the sequence segment.
Reference to the expression “3′” end region of a sequence segment\” there is intended that the localization of the sequence of nucleotides referred to is towards the 3′ terminal end of the sequence segment.
The term “amplicon” as used herein refers to a polynucleotide DNA or RNA molecule that is the product of an enzymatic or chemical-based amplification event or reaction. An amplicon may be single or double stranded. Enzymatic or chemical-based amplification events or reactions include, without limitation, the polymerase chain reaction (PCR), loop mediated isothermal amplification, rolling circle amplification, nucleic acid sequence base amplification, and ligase chain reaction or recombinase polymerase amplification.
The term “primer” or “primer polynucleotide”, as used herein, refers to an oligonucleotide that can hybridize to a template nucleic acid and permit chain extension or elongation using a nucleotide incorporating biocatalyst. A primer nucleic acid that is at least partially complementary to a subsequence of a template nucleic acid is typically sufficient to hybridize with the template nucleic acid for extension to occur. primer nucleic acid can be labeled, if desired, by incorporating a label detectable by radiological, spectroscopic, photochemical, biochemical, immunochemical, or chemical techniques.
The term, “extended primer”, as used herein, refers to a primer to which one or more additional nucleotides have been added. “Primer extension” is the action of the enzyme by which additional nucleotides are added to the primer.
The term “complementary”, as used herein, refers to the topological compatibility or matching together of interacting surfaces of a probe molecule, such as a primer, and its target. Thus, the target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
The term “hybridization” refers to a process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.
The term “anneal” refers to the process by which a single-stranded nucleic acid sequence pairs by hydrogen bonds to a complementary sequence, forming a double-stranded nucleic acid sequence, including the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured)
The term “subject”, as used herein, refers is to an individual. Non-limiting examples of a subject may include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject may be a mammal such as a primate or a human.
In one example, an “adaptor” is a single stranded DNA. The adaptors are versatile as their sequence and length can be changed for various applications (LAMP, qPCR, bioanalyzer . . . ) and can have moieties attached to their 3′ and 5′ ends for other detection modalities (DTECT-Fluo).
The term “detectable label”, as used herein, refers to a composition that when linked to a molecule of interest renders the latter detectable, via, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels may include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
The term “next generation sequencing” (NGS) includes any form of high-throughput DNA or RNA sequencing. This includes, without limitation, sequencing by synthesis, sequencing by ligation, nanopore sequencing, single-molecule real-time sequencing and ion semiconductor sequencing.
In some aspects, there is provided a method of detecting a mutation in a target polynucleotide in a sample from a subject.
Accordingly, the methods herein may be used in the detection or identification of such polynucleotide mutations which may be indicate the presence or absence of a particular mutation, sequence variation, or polymorphism.
Polymorphisms include both naturally occurring, somatic sequence variations and those arising from mutation.
In some examples, there is provided methods for the identification of mutations in a target polynucleotide for identifying mutations associated with disease and/or markers thereof.
In some examples, there is provided methods for the identification of mutations in a target polynucleotide in microorganism(s), including but not limited to, bacteria, fungi, protozoa, ciliates, and viruses. The microorganisms are not limited to a particular genus, species, strain, or serotype.
In some examples, there is provided methods for the identification of a mutation in a target polynucleotide from a sample for rapid and accurate identification of sequence variations that are genetic markers of disease, which can be used to diagnose or determine the prognosis of a disease.
The identification of these “disease” markers is dependent on the ability to detect changes in genomic markers in order to identify errant genes or polymorphisms. Genomic markers (all genetic loci including single nucleotide polymorphisms (SNPs), microsatellites and other noncoding genomic regions, tandem repeats, introns and exons) can be used for the identification of all organisms, including humans. These markers provide a way to not only identify populations but also allow stratification of populations according to their response to disease, drug treatment, resistance to environmental agents, and other factors.
Diseases characterized by genetic markers can include, but are not limited to, atherosclerosis, obesity, diabetes, autoimmune disorders, and cancer.
The term “cancer”, as used herein, refers to a variety of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as “cancer cells”, possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain typical morphological features. Cancer cells may be in the form of a tumour, but such cells may also exist alone within a subject, or may be a non-tumorigenic cancer cell. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.
Accordingly, the identification of mutations in a target polynucleotide in a sample from a subject may be used in applications, including but not limited to, oncology diagnostics, animal breeding, precision genetic editing applications,—including but not limited to base editing, prime editing, CRISPR, in laboratory animal models or plants/crops.
As used herein, “sample” or “biological sample” refers to a composition containing a material to be detected, such as a target polynucleotide.
In some examples, “sample” or “biological sample” refers to materials obtained from or derived from a subject or patient. A sample or biological sample includes sections of tissues such as biopsy (e.g., tumor biopsy) and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, circulating tumor cells, and the like), lymph, sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc.
In some examples, a biological sample may be from a sample from a nasal swab, a sample from an oropharyngeal swab, a sputum sample, a lower respiratory tract aspirate, a bronchoalveolar lavage, a nasopharyngeal wash/aspirate or a nasal aspirate.
In other examples, “sample” or “biological” sample may refer to any material obtained from, for example, an animal such as a human or other mammal, a plant, a bacterium, a fungus, a protist or a virus.
Methods for obtaining a sample or biological sample are known.
In some examples, the sample is from a eukaryote, a prokaryote, or a viruses.
In some examples, the sample is from a mammal, a plant, a bacterium, a fungus, a protest, or a virus.
In a specific example, the subject is a human.
In one aspect there is provided a method of detecting a mutation in a target sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotide sample, the method comprising:
In one example, the ligase is a T4 ligase.
In one example, the T4 ligase is a heat resistant (Hi-T4) T4 ligase, a salt-tolerant (Salt-T4) T4 ligase or, a highly concentrated (T4-HC) T4 ligase.
In one example, the reaction temperature is between about 16° C. and about 37° C.
In one example, the reaction temperature is between about room temperature.
In one example, the reaction time is about 10 min or less than 10 min.
In one aspect there is provided a method of detecting a mutation in a target polynucleotide in a sample from a subject, the method comprising:
In one example, step b) further comprises a competitor DNA.
In one example, the concentration of the competitor DNA is about 1 pmol.
In one example, the ligase is a T4 ligase.
In one example, the T4 ligase is a heat resistant (Hi-T4) T4 ligase, a salt-tolerant (Salt-T4) T4 ligase or, a highly concentrated (T4-HC) T4 ligase.
In one example, the reaction temperature is between about 16° C. and about 37° C.
In one example, the reaction temperature is about room temperature.
In one example, the reaction time is about 10 min or less than 10 min.
In one example, the sample is from a eukaryote, a prokaryote, or a virus.
In one example, the subject is a mammal, a plant, a bacterium, a fungus, a protest, or a virus.
In one example, the sample is isolated from a cell, a cell pellet, a cell extract, a tissue, a biopsy, or biological fluid, obtained from the subject
In one example, the target polynucleotide is the PIK3R1 gene.
In one example, the sample is from a cancer sample.
In one example, the sample is from a nasal swab, a sample from an oropharyngeal swab, a sputum sample, a lower respiratory tract aspirate, a bronchoalveolar lavage, a nasopharyngeal wash/aspirate or a nasal aspirate.
In one example, the subject is a human.
The term “about”, as used herein, when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.
Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.
We have previously established Dinucleotide signaTurE CapTure (DTECT) (
Here, we demonstrate that DTECT captures SARS-CoV-2 variant signatures from different strains with high efficiency and specificity.
We describe herein an enhanced version of DTECT that we validate on emerging SARS-CoV-2 variants of concern (VOC). The improvements described herein limit the number of steps, hands-on time to derive a 10 min single-step one-pot signature capture. These experiments allow direct implementation of DTECT into standard SARS-CoV-2 diagnostics to detect variants of concern and expand the possibilities to apply DTECT for basic laboratory experiments, such as CRISPR-based precision genome editing.
DTECT relies on two successive enzymatic reactions 1) digestion using a type IIS restriction endonuclease to expose genetic signatures and 2) ligation of DNA adaptors complementary to the signatures using a DNA ligase to capture signatures (
DTECT Captures SARS-CoV-2 Variant Signatures with High Specificity
Given the high versatility of DTECT to identify all types of dinucleotide signatures, we tested whether DTECT can capture SARS-CoV-2 genetic signatures. Emerging circulating strains of SARS-CoV-2 (e.g., B.1.17, B.1.351, and P.1 lineages) carry multiple genomic mutations of concern which increase transmissibility and partially prevent recognition by antibodies (e.g., del69-70, K417N, K417T, E484K, and N501Y). We designed Acu1-tagging primers to capture various SARS-CoV-2 signatures from the SARS-CoV-2 reference sequence38. Using synthetic DNA molecules that encode various mutations, we determined that DTECT captures each signature of the SARS-CoV-2 reference sequence with high sensitivity and specificity, as the emerging variants were not detected (
DTECT required about 4-5 hours to execute and requires multi-step procedures, which is not optimal for routine variant detection. In addition, they do not facilitate the execution of basic laboratory experiments.
Therefore, we evaluated DTECT for its performance on ligation efficiency (referred to as capture score) and specificity (specificity score).
DTECT utilizes two sequential enzymatic reactions to capture specific signatures. First, the type IIS restriction enzyme Acu1 digests a genomic amplicon to generate a 3′ dinucleotide overhang. Second, a DNA ligase ligates specific DNA adaptors complementary to either the reference or variant signatures. Finally, an isolation step that separates the two enzymatic activities serves to isolate one of the two DNA fragments. This step helps to preferentially ligate the adaptor without reassembling the two DNA fragments generated at the digestion step, thereby enabling high precision ligation.
To confirm that these three steps (Acu1 digestion, beads isolation, and adaptor ligation) are critical to DTECT, we tested the original DTECT (also referred to as DTECT1.0) with or without Acu1, the isolation beads step, or DNA ligase (
Surprisingly, omitting the beads isolation step affected neither capture efficiency nor specificity (
An analysis of the product of ligation by analytic PCR confirmed the expected product (
These results indicate that the beads isolation step is dispensable, which not only decreases the overall cost of DTECT but also facilitates the implementation of an improved method with the minimal requirement for off-the-shelves enzymes.
Altogether, these results demonstrate that the sensitivity and specificity of DTECT1.0 can be improved.
These data prompted us to reassess each condition of DTECT1.0 to develop an optimized and accelerated signature capture with enhanced capture efficiency and specificity.
In the DTECT1.0 protocol, 1.25 units of Acu1 digest 0.2 pmol of Acu1-tagged amplicon during 60 minutes at 37° C.
We conducted a time-course experiment ranging between 10 seconds and 60 minutes to determine the optimal digestion conditions.
Surprisingly, we observed that the digestion of 0.2 pmol of DNA with 1.25 units of Acu1 is rapid. In some examples, the digestion is considered to be substantially instantaneous (
A 10-second incubation leads to complete capture, comparable to a 60 min digestion (
Inactivation of Acu1 by incubating Acu1 at 65° C. for 20 min, is suggested by the suppliers to limit potential interference between Acu1 and ligation.
To test whether Acu1 heat inactivation can be accelerated, we conducted a time-course experiment (
Next, we decided to test how the ligation reaction can be optimized to improve capture efficiency and specificity. The quantity of substrates (adaptors) and enzyme concentration (DNA ligase) influence DNA ligation efficiency. Therefore, we titrated the concentration of DNA ligase (
Altogether, we determined the optimal conditions for high efficiency and specificity capture. These changes include removing the beads isolation step and implementing rapid digestion and ligation reactions. These enhancements result in a 92% decrease in the duration of the capture, unlocking a rapid (12 min) and sensitive signature capture, referred to as DTECT2.0 (
To test the compatibility of Acu1 and ligase, we combined in a single pot but two independent steps: digestion and ligation reactions. First, Acu1 was added with the DNA and incubated for 2 min (1 min digestion at 37° C. and 1 min denaturation at 65° C.). Then, DNA ligase and adaptors were added for 10 min at 25° C. Ligation reactions were immediately stopped by heating the reaction for 10 min to ensure the quantification of the capture is not affected by subsequent handling of the samples. Remarkably, this single pot experiment led to a robust and specific capture (
Our objective is to induce the capture in a single-step in which Acu1 and T4 ligase activities function simultaneously at room temperature. This approach can only be successful if minimal Acu1 activity is provided to limit further digestion of the adaptor ligation reaction. We titrated the amount of Acu1 and measured its activity at 25° C. Interestingly, we find that a dilution of Acu1 to 0.5 units leads to optimal capture, comparable with the reference (
With the demonstration that digestion and ligation can be incubated in a single tube in optimized conditions, we tested whether the single pot isothermal incubation of the digestion and ligation for 10 min at room temperature. Strikingly, we found that signature capture is efficient even without Acu1 inactivation, demonstrating that the ligation is not affected by the presence of minimal Acu1 activity (
Finally, we tested whether direct dilution of the Acu1-tagged amplicon or various purification protocols, including gel purification, column purification, or beads purification are compatible with the single-step digestion/ligation. We observed that the purification of Acu1-tagged amplicon is dispensable as purification or dilution of the PCR leads to comparable results (data not shown). Notably, a large window of dilution enables high-efficiency capture without losing specificity. This data suggests that the Acu1-tagged amplicon does not require purification but also that the performance of DTECT2.0 will not be affected by the PCR efficiency or the quantity of starting material. These data establish an improved method for the highly sensitive single-step capture of genomic signatures.
In this work, we developed a new capture of genomic signatures for the straightforward detection of SARS-CoV-2 variants. The development of a single-step single-pot DTECT is particularly useful for precision genome editing applications because it will help democratize DTECT for basic research labs.
Alternative detection methods include sequencing technologies, such as next-generation sequencing or Sanger sequencing. However, these approaches are expensive, have a considerable turnaround time of several days (Sanger sequencing) to weeks (NGS) (compared to a few hours for DTECT), and require the involvement of third parties. On the other hand, DTECT2.0 as described herein is accessible because it only requires off-the-shelf reagents (e.g., T4 ligase and Acu1), which are available from various suppliers, and minimal equipment (e.g., thermocycler and qPCR). In addition, an advantage of DTECT2.0 is that it uses a standard library of 16 adaptors to detect each possible dinucleotide signature. Thus, DTECT2.0 offers significant advantages over approaches utilizing sequencing technologies for the rapid monitoring of variants. For instance, DTECT2.0 identifies all variant types by capturing targeted signatures with a unique library of adaptors and achieves high specificity and sensitivity detection of molecular signatures through a strong covalent ligation (i.e., capture). Moreover, multiple analysis modalities can be derived to analyze the ligated product(s) as a signal for the presence of variants in patient specimens.
DTECT2.0 is a robust molecular diagnostic tool with several significant features that makes it more reliable, specific, and efficient than other rapid diagnostic tests that utilize mutation-specific PCR primers and probes to identify variants26-28 Indeed, these methods have a low specificity conferred by a single nucleotide mismatch to differentiate a variant from the reference (e.g., a 25 nt probe/primer: 1/25 nt→4% specificity target). In contrast, DTECT relies on a dinucleotide capture to differentiate the variant from the reference (½ nt→50% difference in the target), resulting in a strong specificity. Additionally, DTECT is a ligation-based approach that generates covalent phosphodiester bonds between signatures and adaptors, creating stable ligation products, unlike primers/probes approaches which rely on weak and transient nucleic acid interactions. The production of a stable ligated product allows the deployment of multiple modalities to analyze the captured material, as proposed below. Thus, DTECT is also particularly relevant for clinical applications. Indeed, DTECT provides robust internal controls in all SARS-CoV-2 positive samples because it must always detect either the WT or the variant SARS-CoV-2 signatures. Moreover, each variant can be detected using four independent signatures (2 flanking Acu1-tagging primers from each DNA strand), providing rigorous validations required to deliver high-confidence clinical results. Finally, DTECT is a robust qualitative and quantitative approach with limited technical variabilities because it exploits a unique couple of qPCR oligo pair to analyze the ligation products (
An appealing advantage of DTECT for further improvements is the flexibility of the adaptors and 5′-end of the Acu1-tagging oligos. For instance, 5′- and 3′-ends of the adaptors are available for the addition of dyes/quenchers, and the modifications in the DNA sequences do not affect DTECT efficiency.
Loop-mediated isothermal amplification (LAMP) is a sequence-specific isothermal DNA amplification method that produces a large quantity of DNA39. The rapid production of DNA modifies the pH, which induces a change in the color of pH-sensitive dyes40 that can be visualized by the naked eye or under blue/UV light. We will couple DTECT with LAMP by integrating the LAMP-specific sequences into the adaptors and 5′ sequence of the Acu1-tagging primers. Therefore, upon ligation of the signatures to the adaptors, the LAMP sequences will be reconnected, generating an amplification signal that can be visualized in real-time. To optimize DTECT-LAMP, multiple color dyes such as calcein, hydroxynaphthol blue, SYBR green I, berberine and EvaGreen40,41 may be used By coupling our optimized single-step DTECT (<10 min) with LAMP (˜15-30 min), we expect to achieve visual detection of SARS-CoV-2 variants in <1 hr without instrumentation.
In another example, a quencher may be added (e.g., Iowa BlackFQ) and a fluorescent dye (e.g., 6-carboxyfluorescein) to the 5′- and 3′-ends of the Acu1-tagging oligo and adaptors. Various commercially available quenchers and dyes may either be placed at 5′- or 3′-end of the Acu1-tagging oligo and adaptors to determine the best combination for efficient and multiplexed signal detection. Upon successful covalent linkage induced by ligation of the adaptors to the complementary signature, the quencher will block fluorescence emission, resulting in a loss of fluorescence over time, as easily detectable with a transilluminator or a fluorescence plate reader42. Multiple adaptors with different dyes may be used to recognize various variant signatures will unlock multiplexed detection of variants. DTECT-Fluo will provide an all-in-one multiplexed detection of variants, in which all components are present (digestion, ligation, and detection) for real-time detection (<5 min total) without experimenter intervention.
Synthetic DNA molecules containing portions of the SARS-CoV-2 genome with or without mutations were purchased as gBLOCK DNA fragments (IDT). The DNA fragments were resuspended in TE buffer, cloned into the pCR-Blunt II-TOPO vector (ThermoFisher Scientific), and transformed into DH5a. Successful cloning and SARS-CoV-2 sequence were confirmed by Sanger sequencing.
The same library of adaptors is used for the capture of 16 dinucleotide signatures. The library comprises 16 double-stranded DNA adaptors generated from 17 individual oligonucleotides (sequences available in table 1). It contains one constant oligonucleotide (named OB1), which contains a sequence at the 3′ end (5′-gaattcgagctcggtacccg-3′)(SEQ ID NO: 86) for the detection of the ligated products, and 16 individual oligonucleotides, which are composed of a sequence complementary to the constant oligonucleotide and one of the 16 different dinucleotides at their 3′ end (named OB2-OB17).
Each oligonucleotide is resuspended at a concentration of 100 μM in TE (10 mM Tris and 0.5 mM EDTA). The annealing reactions are composed of 2.5 μl of the constant oligonucleotide, 2.5 μl of each unique dinucleotide oligonucleotide, and 1× ligase buffer. The reactions are incubated for 5 min at 95° C. to remove any potential secondary structures followed by a gradual temperature decrease from 95° C. to 15° C. at a ramp rate of 1° C./s. Then, 100 μl H2O is added to dilute the adaptors at 5 uM. Adaptors are stored at −20° C. or −80° C.
The Acu1-tagging PCR utilizes a pair of primer named “Acu1-tagging primer” and “reverse primer”. The objective of the Acu1-tagging PCR is to insert an Acu1 motif 14 bp upstream from a targeted dinucleotide, introduce a handle that is used for the detection, and amplify the locus of interest.
The Acu1-tagging primers is a 60 nt long oligonucleotide that contains an Acu1 motif (5′-CTGAAG-3′) as a hairpin 14 np from the 3′ end of the primer. In addition, it also contains a non-complementary handle sequence of 25 nt (5′-GCAATTCCTCACGAGACCCGTCCTG-3′) (SEQ ID NO: 53) that is used for the detection. Therefore, the Acu1 tagging primer has the following architecture: 5′-N(15)CTGAAGN(14)-3′ (SEQ ID NO: 54) with “N” corresponding to A, T, G, or C bases complementary to the targeted locus.
The reverse primer is designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) with a length of “min=25, Opt=27, Max=30” and a Tm of “min=57.0° C., opt=60.0° C., max=63.0° C.”
The Acu1-tagging PCR is performed in a 25 μl with 1 unit Q5 polymerase as recommended (NEB), 1× Q5 buffer, 1 μM of each primer, 10 ng plasmid template, 0.1 mM dNTP in a thermocycler: 95° C. for 30 s; 40 cycles of 95° C. for 10 s, 58° C. for 10 s, 72° C. for 45s and a final amplification at 72° C. for 1 min. The PCR reaction is loaded on a 2% agarose gel in TAE buffer, and the amplicon is extracted from gel and column purified (Zymo Research #D4008). The purified Acu1-tagged amplicon is quantified with the nanodrop 2000 and stored at −20° C.
The original DTECT protocol has been conducted as detailed previously33 Briefly, DTECT relies on the amplification of the genomic locus of interest using an Acu1-tagging primer. The purified Acu1 tagged amplicon is digested by Acu1 in a 20 μl reaction as follows: 0.2 pmol Acu1 tagged amplicon, 1.25 units Acu1 (NEB #0641) in 1× CutSmart buffer. The digestion is incubated at 37° C. for 1 hour followed by heat inactivation at 65° C. for 20 min. SPRI beads separate the digested fragments by mixing beads at a ratio of 1:1.8 of Agencourt AMPure XP magnetic beads. 10 μl of digestion is mixed with 18 μl of beads by pipetting up and down ten times and incubated at room temperature for 5 min. The tube is then placed on a magnetic rack, and the supernatant is recovered and diluted in 40 μl H2O. Next, the ligation of the adaptors is performed in the following reaction: 6.5 μl H2O, 2 μl of 5× ligase buffer, 0.5 μl T4 ligase (ThermoFisher Scientific), 0.5 μl adaptor, and 0.5 μl of the purified digested product. The ligation reaction is incubated for 1 hour at 25° C. in a thermocycler. The reaction was then stopped by incubating the reaction at 65° C. for 10 min to denature the ligase. The captured material was detected either using quantitative PCR or analytical PCR.
The qPCR is conducted using the QuantStudio 6 (Applied Biosystems). qPCR reactions were performed as follows: 5 μl of 2× SYBR Green master mix, 0.1 μl of primer OB1 (100 μM), 0.1 μl of primer OB2 (100 μM) and 1 μl of ligated products in a 10 μl reaction. The qPCR program is the following: 1) A hold stage of 1 cycle at 50.0° C. for 2 min and 95.0° C. for 10 min. 2) A PCR stage of 40 cycles at 95° C. for 10 seconds and 60° C. for 30 seconds. 3) A melt curve stage of 1 cycle of incubations at 95° C. for 15 seconds, 60° C. for 1 min, and 95° C. for 15 seconds. The quantification of the captured material (capture score) and the difference between the specific and non-specific adaptor (specificity score) are calculated as described below.
The analytical detection is performed by standard Q5 PCR in a 12.5 μl containing 0.1 μl Q5 polymerase, 1× Q5 buffer, 0.5 μM OB18, 0.5 μl OB19, 0.05 mM dNTP, and 1 μl ligation products. The PCR program (Proflex 3×32) for the analytical reaction is the following: 95° C. for 1 min and 22 cycles of 95° C. for 10 s, 65° C. for 5 s and 72° C. for 7 s. The PCR reaction was incubated with SYBR Gold (ThermoFisher Scientific), loading dye, and loaded on a 2% agarose gel with TAE buffer.
The experiment without bead isolation was carried out following the DTECT1.0 procedure, but the bead step was omitted. Without the beads step, the digestion reaction was diluted by adding 100 μl of H2O. This dilution was subsequently used in regular ligation. In addition, enzymes have been diluted in their working buffer, such that Acu1 was diluted in 1× Cutsmart buffer and T4 ligase was diluted in 1× ligase buffer. All reactions were conducted in independent duplicates. All incubations were conducted in a thermocycler.
The DTECT2.0 protocol relies on DTECT1.0 but includes several optimizations. For example, the duration of the digestion/inactivation has been shortened, a dilution in H2O has replaced the bead isolation step, and the adaptor ligation step has been shortened.
The Acu1-tagging PCRs are conducted as described above. The Acu1 digestion/inactivation is performed in 20 μl by mixing 0.2 pmol of Acu1-tagged amplicon with 1.25 units Acu1 in 1× Cutsmart buffer. The digestion is incubated at 37° C. for 1 min followed by 1 min at 65° C. The digested reaction is diluted by the addition of 100 μl H2O and used directly for the ligation. The adaptor ligation is conducted in 10 μl by mixing 2 μl of ligase buffer, 0.5 μl T4 ligase (Invitrogen), 0.5 μl of the selected adaptor, and 0.5 μl diluted digestion. The reaction is incubated for 10 min at 25° C. The reaction is stopped by incubating 10 min at 65° C. Finally, analytical or quantitative PCR is performed as detailed above.
The one-pot DTECT2.0 protocol merges DNA ligation and Acu1 digestion in a single tube. It utilizes an optimized quantity of Acu1-tagged amplicon compatible with the one-pot digestion-ligation reaction. The Acu1-tagging PCRs are conducted as described above. The reactions are conducted in a single tube but separated in two independent steps as follows: 0.005 pmol of Acu1 tagged amplicon is digested in a 7 μl reaction by mixing 1 μl Cutsmart buffer, 1.25 μl of diluted Acu1 (Acu1 was diluted 1/10th in 1× Cutsmart buffer) and completed with H2O. The digestion is incubated for 1 min at 37° C. and 1 min at 65° C. in a thermocycler. Then, 2 μl ligase buffer, 0.5 μl of selected adaptor and 0.5 μl T4 ligase (Invitrogen) are added to the reaction and incubated for 10 min at 25° C. The ligation was stopped by incubation at 65° C. for 10 min. Finally, analytical or quantitative PCR is performed as detailed above.
A standard curve to determine the efficiency of the qPCR amplification and the linearity of the amplification was generated with a plasmid that contains a DTECT ligation product (Addgene #139333) using primers OB18 and OB19 (sequences in Table 1). The linearity of the standard curve has the mathematical formula: y=−3.3245×+7.5504.
Each sample analyzed by qPCR is tested in technical duplicates, and the mean Ct for each sample is calculated. The capture score is defined as the concentration of the captured material for each sample multiplied by 10{circumflex over ( )}6. It is measured as follow: Capture score=(10{circumflex over ( )}[(Mean Ct−7.5504)/−3.3245])×10{circumflex over ( )}6. The reported capture score corresponds to the mean of two independent experiments and is shown as LOG10.
The efficiency score corresponds to the absolute value of the difference in Ct between the specific and non-specific adaptors. It was calculated using the formula: “=ABS(Ct specific adaptor−Ct non-specific adaptor)”. Thus, the reported efficiency score corresponds to the mean of two independent experiments.
Sequencing technologies, such as Sanger or Next-Generation Sequencing, are the most common methods to detect genomic sequences and variants of interest. Despite their high accuracy, these technologies remain time-consuming and expensive. There is currently no rapid and cost-efficient method that can be efficiently conducted using all-in-one reactions to detect desired genetic signatures. Here, we establish a platform for rapid and accurate capture of genetic signatures, named Dinucleotide signaTurE CapTure version 3 (DTECTv3). We develop and optimize an all-in-one reaction to rapidly capture desired genetic changes that can be prepared from off-the-shelf reagents and using a set of premade adaptors. We also derive multiple detection modalities with complementary strengths for applications requiring quantitative, qualitative, or visual detection of genetic mutations. We apply DTECTv3 to accurately quantify various mutation types, including transition and transversion mutations, small insertions, and deletions from SARS-CoV-2 variants of concern, cancer mutations, or introduced by cutting-edge CRISPR technologies such as base editing and prime editing. DTECTv3 expedites the accurate detection of genetic signatures for routine laboratory experiments for a fraction of a dollar and enriches the toolkit of the detection methods for CRISPR-based precision genome editing.
Identifying variations in DNA sequences is a routine task in basic research for genetic testing, clinical diagnostic, or forensic purposes. Over the past decades, several technologies utilizing Sanger or Next-Generation Sequencing (NGS) platforms have been developed to facilitate the sequencing of DNA molecules, enabling the determination of DNA sequences and the identification of variants. However, although these technologies are easily accessible, as companies offer genomic platforms and sample processing, they remain time-consuming (several days to weeks) and expensive (from several dollars to thousand dollars) for routine laboratory experiments. Moreover, it requires the processing of samples by third parties, which can cause errors and contaminations during sample manipulation. The past two decades have also witnessed the development of an accelerated number of new techniques using variant-specific primers or probes. However, these techniques are not robust because the efficacy and specificity of the detection are strongly dependent on the sequence and mutation. In addition, these approaches lack specificities as they rely on weak and transient nucleic acid interactions to distinguish between genetic variants, which often differ from the reference by only one nucleotide.
The recent democratization of CRISPR-based precision genome editing technologies, including base editing and prime editing, has increased the need for rapid, sensitive, and accurate methods to detect genetic signatures in routine laboratory settings. These novel genome editing technologies are revolutionizing the modeling and correction of variants in cellular and animal models. In particular, base editing introduces transition mutations, and prime editing inserts all types and combinations of genetic changes (transitions, transversions, deletions, and insertions). Therefore, a detection method that can rapidly and easily identify all types of mutations is highly desirable.
The advent of recombinant DNA technology has revolutionized molecular biology by enabling the assembly of novel DNA sequences. The discovery of DNA ligases and restriction enzymes has emulated this revolution by allowing precise cutting and assembly of DNA fragments with partial overlap sequences. In particular, enzymes from the type IIS (S for “shifted”) family have the unique particularity to cut DNA at a shifted but precise distance from their recognition motif, which allows the cleavage of unknown sequences, enabling the development of a variety of applications. Recent advances in molecular cloning approaches, such as Gibson Assembly, have facilitated the assembly of high complexity DNA fragments. The coordinated action of multiple enzymes in an all-in-one ready-to-use reaction has helped democratize these approaches for the complex manipulation of DNA fragments. However, despite the importance of detecting genetic sequences and associated mutations, there are still no all-in-one assays available for rapid detection of genetic signatures for routine laboratory experiments.
Here we present DTECTv3, a sequencing-free method that leverages two enzymatic activities to simultaneously expose and capture genetic signatures of interest. We unlock rapid concomitant single-step digestion-ligation of signatures by adding competitor DNA fragments that inhibit Acu1 to prevent the digestion of the ligated adaptors and by enhancing the enzymatic reaction to accommodate the two enzymes. Importantly, DTECTv3 only requires a library of 16 premade and adjustable adaptors to capture all possible types of genetic changes. We illustrate this versatility of the adaptors by unlocking rapid isothermal visual detection of variants, which extends the possibilities of this platform beyond quantitative and qualitative detection. We also show that DTECTv3 enables the rapid detection of emerging SARS-CoV-2 variants of concern and various mutation types, including transition, transversion, small insertions, and deletions, introduced by base editing and prime editing. This platform which utilizes an all-in-one capture reaction with premade adaptors will facilitate and accelerate the routine detection of genetic signatures, including genetic changes introduced by genome editing technologies
In previous studies, we have established a technique to detect genetic signatures, called Dinucleotide signaTurE CapTure (DTECT), that leverages two enzymatic activities: a type IIS restriction endonuclease that is programmed to expose targeted genetic signatures of interest and a DNA ligase that attaches DNA adaptors to enable signature detection (
The locus of interest is first amplified by PCR using an Acu1-tagging primer that contains a small hairpin to introduce a six-nucleotide motif (5′-CTGAAG-3′) recognized by the Type IIS enzyme Acu1 (
We previously demonstrated that DTECT could readily identify cancer mutations in the bone marrow of cancer patients and for precision genome editing in cell lines, organoids, and animal tissues. The COVID-19 pandemic has illustrated the need for easy-to-conduct and rapid methods for detecting genetic signatures. Strains of SARS-CoV-2 have emerged (e.g., alpha, beta, gamma, and delta variants) with multiple mutations (e.g., K417N, K417T, E484K, and N501Y), which are unique in the different SARS-CoV-2 lineages. For example, K417N and K417T are specific to the beta and gamma variants. These variants increase transmissibility and partially prevent recognition by vaccine-induced antibodies. Given the high versatility of DTECT in identifying all types of dinucleotide signatures, we tested whether the original DTECT (also referred to as DTECTv1) (
Although DTECTv1 is robust and rapid to execute (˜4-5 hours), it requires two independent enzymatic reactions (digestion and ligation steps, as shown in
DTECT utilizes two sequential enzymatic reactions, a restriction digestion (
To test whether these three independent steps (Acu1 digestion, beads isolation, and adaptor ligation) are essential for DTECT, we conducted DTECTv1 to capture the SARS-CoV-2 E484K variant, but we independently omitted each step/enzyme (i.e., Acu1, beads, or ligase) (
These data prompted us to reassess each step of DTECTv1 to develop an optimized capture. First, we tested how parameters influence digestion by Acu1. In the DTECTv1 protocol, Acu1 digests the Acu1-tagged amplicon for 60 minutes at 37° C. as recommended by the enzyme suppliers. We conducted a time-course experiment ranging between 10 seconds and 60 minutes to determine the optimal digestion conditions. Surprisingly, we observed that a 10-second incubation leads to a capture efficiency comparable to a 60 min digestion (
Notably, the Acu1 activity must not persist during adaptor ligation to avoid the digestion of the ligated product (dinucleotide-adaptors) by Acu1. For this reason, Acu1 is inactivated by incubating the reaction at 65° C. for 20 min, as recommended by the Acu1 suppliers. To test whether Acu1 heat inactivation can be accelerated, we initially heat-inactivated Acu1 from 30 seconds to 20 min and then added the Acu1-tagged amplicon to the reaction so that the level of the captured product determines if Acu1 was efficiently inactivated. We restricted the digestion to 1 min at 37° C. because previous experiments revealed that digestion is already completed after 10 seconds (
Next, we tested whether the ligation reaction can be optimized to improve signature capture. We titrated the concentration of DNA ligase and determined the conditions that provide the highest capture efficiency and specificity (
Altogether, these experiments suggest that we can enhance DTECTv1 by removing the beads isolation step and by accelerating Acu1 digestion (from 60 min to 1 min), Acu1 inactivation (from 20 min to 30 s), and adaptor ligation (from 60 min to 1 min). To test whether these changes can be combined and to compare DTECTv1 (3 steps; ˜2 hrs 30 min total capture time) to the accelerated DTECTv2 (2 steps; <5 min capture) (
Removing the bead isolation step makes it possible to envision that the two enzymatic activities (Acu1 digestion and DNA ligation) could be active into an optimized buffer that can accommodate both activities concomitantly.
To test the compatibility of Acu1 and the DNA ligase to work in a single reaction, we conducted a capture in which the two enzymatic reactions are physically separated in two independent steps/tubes in their respective optimal buffers (
Next, we systematically evaluated the activity of several DNA ligases to determine the most effective ligase to capture signatures in a single pot. The T4 ligase showed the most robust capture activity among the different ligases, followed by the T3 ligase (
To enhance the concomitant digestion/ligation, we tested whether the composition of the buffer could increase capture efficiency. We tested how the omission of each component affects the capture efficiency to determine a minimal buffer. We observed that removal of the Acu1 buffer improves capture specificity (
One key characteristic of type IIS enzymes is that they do not cleave their recognition motifs but cut DNA at a shifted distance in the bound DNA. Consequently, type IIS enzymes can remain bound to DNA substrates after digestion, and in the case of the ligation of compatible DNA sequences, Acu1 would digest it, preventing the digestion of the adaptors. We hypothesized that the addition of exogenous DNA fragments that contains an Acu1 motif would limit the capacity of Acu1 to digest newly ligated fragments. To test this, we prepared a double-stranded DNA consisting of an Acu1 motif sequence surrounded by 12 nt to avoid competitor cutting by Acu1. The addition of 1 pmol of the competitor, but not a DNA control that lacks the Acu1 motif, stimulated the capture efficiency (
Altogether, we were able to enforce single pot digestion/ligation despite persisting Acu1 activity by 1) increasing the concentration of adaptors, 2) optimizing buffer conditions, and 3) adding a competitor DNA fragment (
Altogether, these data establish a set of key optimizations that lead to the development of version 3 of DTECT (DTECTv3) for the rapid capture of genomic signatures (
While the previous experiments have established a rapid single pot capture, the purification of Acu1-tagged amplicon limits the throughput and rapidity of the overall protocol. Given the low amount of Acu1-tagged amplicon needed in DTECTv3, we hypothesized that the purification of Acu1-tagged amplicon might be streamlined. We tested whether direct various purification protocols, including gel purification, column purification, or beads purification, are compatible with the single-step digestion/ligation. As a control, direct dilution ( 1/100th) of the Acu1-tagged amplicon was tested. Surprisingly, we observed that the purification of Acu1-tagged amplicon is dispensable as purification or dilution of the PCR leads to comparable capture (
Quantification with DTECTv3 is Accurate and Specific
Next, we tested whether DTECTv3 is accurate and specific for the quantitative detection of genomic signatures. First, we compared whether the 16 dinucleotides are captured with the same efficiency by their respective complementary adaptors. Strikingly, we observed a highly consistent capture between the 16 possible dinucleotides using DTECTv3 (
Second, we tested the accuracy of DTECT to quantify the relative amount of genetic signatures. We mixed various quantities of WT and variant signatures (ratios: 100:0, 75:25, 50:50, 25:75, and 0:100) and captured the respective signatures using DTECTv3. The relative capture frequency was highly quantitative for detecting a mixture of SARS-CoV-2 variants of concerns and a cancer mutation (
Altogether these experiments demonstrate that the simplified and accelerated DTECTv3 protocol is highly accurate, does not suffer from technical variabilities, and precisely quantifies the relative frequency of genetic signatures.
Precision genome editing technologies, such as base editing and prime editing, are revolutionizing genetic studies in cellular and animal models.
We tested DTECTv3 to detect newly generated genetic signatures induced by base editing and prime editing. We transfected HEK293T cells with cytosine base editor (CBE) and guide RNAs (gRNAs) that were designed to introduce a premature STOP codon to inactivate a series of key DNA repair genes (
Next, we utilized prime editing to introduce various mutation types, including transition, transversion, precise small deletion, and insertions. Prime editing is the most recent and exciting precision genome editing technology developed. Prime editing can introduce virtually any small genomic changes as desired. We first tested whether DTECTv3 identifies newly created genetic signatures by prime editing. DTECTv3 readily detected genomic changes induced by prime editing, including a three-nucleotide insertion (insCTT) and a small deletion (del1T) at the HEK3 locus (
We then derived individual clones harboring cancer mutations successfully introduced into the PCNA gene with prime editing. We used qualitative detection of DTECTv3 to determine if cells were homozygous or heterozygous. DTECTv3 accurately determined the genotype of the cells (
These experiments demonstrate that DTECT streamlines the detection of cells edited with base editing and prime editing. DTECT quantifies the relative amount of WT and edited alleles in cellular pools and clones. These experiments illustrate how DTECT can complement NGS or Sanger sequencing, as a quicker and cost-friendly alternative to determine successful editing and genotyping before more complex analysis of the full spectrum of low-frequency edits by NGS or confirmation of genotyping of the clones of interest by Sanger sequencing.
By accelerating and facilitating the detection of precision genome editing, DTECT can positively impact the generation of precisely edited model systems by facilitating the quantification and genotyping of desired genetic changes in which only a PCR on genomic DNA samples is needed before incubating in an all-in-on reaction for 10 min at room temperature to induce signature capture.
One striking advantage of DTECT is that it uses completely customizable adaptors. We hypothesized that by modifying the adaptor sequences, we could envision additional detection modalities of the ligated product.
Loop-mediated isothermal amplification (LAMP) is a sequence-specific isothermal DNA amplification method that produces a large quantity of DNA. The rapid production of DNA modifies the pH, which induces a change in the color of pH-sensitive dyes that can be visualized under blue/UV light. One important limitation of LAMP is that it requires the identification of specific sets of sequences with particular genomic features (distance between sequences, and G/C contents) in the targeted nucleic acid sequence. The mixture of oligonucleotides complementary to the identified target sequences with the Bst DNA polymerase enables rapid exponential nucleic acid amplification at isothermal temperature. The rapid amplification yields a pyrophosphate ion that changes the color of the reaction if a dye, such as calcein, is added in the reaction. LAMP is a rapid and easy visual method to detect the presence of specific nucleic acid sequences. However, LAMP is not efficient at detecting particular variants within the targeted nucleic acid sequence.
We hypothesized that the DTECT adaptor ligation could trigger the reconstitution of the different LAMP oligonucleotide targets, and therefore, loop amplification would start only if the ligation is efficient, meaning if a signature is present, thereby creating a LAMP approach for the detection of genetic variants. To test this hypothesis, we separated in the LAMP sequences between the 5′ end of the Acu1-tagging oligo and adaptors. Consequently, the LAMP targets would only be reconstituted if the ligation is successful, inducing exponential DNA amplification triggering color change. We included F2 and F3 LAMP sequences derived from the SARS-CoV-2 detection in the 5′ end of the Acu1-tagging oligonucleotide, and the F1, B1, B2, and B3 sequences were included in the adaptors (
Altogether, we adapted the widely used LAMP, which only recognizes the presence of nucleic acids, to specifically detect genetic variants. Importantly, we used validated sequence targets so that DTECT-LAMP detects the presence of specific variants and suppresses the biggest limitation of LAMP, which is the need to identify key sequence targets.
Here, we establish and describe the development of a novel platform for the capture of genetic signatures and derive multiple detection modalities enabling qualitative, quantitative, and visual detection.
DTECTv3 only requires the generation of a PCR product (Acu1-tagged amplicon) that amplifies the locus of interest and “tag” the dinucleotide of interest with the Acu1 motif. This PCR can be generated from any source of DNA or reverse-transcribed RNA and requires little starting material. We note that the generation of a PCR product is also a required initial step for the detection by Sanger sequencing or NGS. The PCR amplicon is then incubated in an all-in-one reaction for 10 minutes room at temperature to expose (i.e., digestion) and capture (i.e., ligation) genetic signatures of interest using a library of adaptors. Finally, the ligated product is detected using three possible detection modalities: qualitative or quantitative PCRs or direct visual detection by loop amplification. Notably, a unique advantage of this platform is that the detection utilizes standard oligonucleotides to detect all genetic variants, mutation types, or genomic loci. This is an important advantage as it limits technical variabilities. Consequently, the use of DTECTv3 is facilitated by the use of common all-in-one master mix reactions for the capture and the detection. These all-in-one reactions contain all the required components to capture, and to detect the ligated product through quantitative PCR, analytical PCR, or DTECT-LAMP. Importantly, all reagents and enzymes necessary to build DTECTv3 are commercially available from multiple suppliers, and the master mixes can be stored for extended periods in a freezer. Altogether, the development of streamlined protocols for the accurate detection of genetic signatures dramatically simplifies the detection of genetic sequences of interest for routine laboratory experiments.
To enable concomitant digestion and ligation in a single reaction, we needed to force the ligation and limit Acu1 activity. We solved this problem on three fronts. First, we optimized the conditions of the reaction to enable simultaneous activities. Second, we forced the ligation by increasing the concentration of adaptors. Third, we added a short DNA fragment containing an Acu1 motif to control Acu1 activity. It is possible that upon DNA cleavage, a structural change is induced in Acu1 to facilitate its release from the DNA while stabilizing its interaction if no cleavage occurred. It remains unclear if this can be generalized to other Type IIS enzymes, as they might have different DNA binding kinetics and interactions.
Alternative detection methods include sequencing technologies, such as next-generation sequencing or Sanger sequencing. However, these approaches are expensive, have a considerable turnaround time of several days (Sanger sequencing) to weeks (NGS) (compared to a few hours for DTECT), and require the involvement of third parties. On the other hand, DTECT is accessible because it only requires off-the-shelf reagents (e.g., T4 ligase and Acu1), available from various suppliers, and minimal equipment (e.g., thermocycler and qPCR). In addition, a critical advantage of DTECT is that it uses a standard library of 16 adaptors to detect each possible dinucleotide signature. Thus, DTECT offers significant advantages over approaches utilizing sequencing technologies to rapidly monitor variants. For instance, DTECT identifies all variant types by capturing targeted signatures with a unique library of adaptors and achieves high specificity and sensitivity detection of molecular signatures through a strong covalent ligation (i.e., capture).
DTECT is a robust molecular diagnostic tool with several significant features that makes it more reliable, specific, and efficient than other rapid diagnostic tests that utilize mutation-specific PCR primers and probes to identify variants. Indeed, these methods have a low specificity conferred by a single nucleotide mismatch to differentiate a variant from the reference (e.g., a 25 nt probe/primer: 1/25 nt→4% specificity target). In contrast, DTECT relies on a dinucleotide capture to differentiate the variant from the reference (½ nt→50% difference in the target), resulting in a strong specificity.
DTECT is highly accurate as it is a ligation-based approach that generates covalent phosphodiester bonds between signatures and adaptors, creating stable ligation products, unlike primers/probes approaches which rely on weak and transient nucleic acid interactions. The production of a stable ligated product allowed the development of multiple modalities to analyze the captured material. Moreover, DTECT provides robust internal controls because, in control samples, the WT but not the variant signatures must be detected. Therefore, the capture of the WT signature acts as a positive control, and the capture of the variant signature provides the background capture. Furthermore, each variant can be detected using four independent signatures (2 flanking Acu1-tagging primers from each DNA strand), providing rigorous validations required to deliver high-confidence results for specific applications. Finally, DTECT is a robust qualitative and quantitative approach with limited technical variabilities because it exploits a unique couple of qPCR oligo pairs to analyze the ligation products (
One of the most appealing advantages of DTECT for further improvements is the flexibility of the adaptors and 5′-end of the Acu1-tagging oligos. For instance, 5′- and 3′-ends of the adaptors are available to add dyes/quenchers, and the modifications in the DNA sequences do not affect DTECT efficiency. Here, we have developed alternative and independent detection approaches. We have successfully coupled DTECT with LAMP by integrating the LAMP-specific sequences into the adaptors and the 5′ sequence of the Acu1-tagging primers. Therefore, upon ligation of the digested product to the adaptors, the LAMP sequences are reconnected, enabling a loop amplification signal to be visualized in real-time. By coupling our optimized single-step DTECTv3 with LAMP, we visually detect variants of interest, including SARS-CoV-2 variants of concern, in ˜40 min (10 min capture+30 min LAMP). DTECTv3-LAMP is very specific as loop amplification occurs specifically in the conditions in which the ligation has been successful.
Synthetic DNA molecules containing portions of the SARS-CoV-2 genome with or without mutations were purchased as gBLOCK DNA fragments (IDT). The DNA fragments were resuspended in TE buffer, cloned into the pCR-Blunt II-TOPO vector (ThermoFisher Scientific), and transformed into DH5a. Successful cloning and SARS-CoV-2 sequence were confirmed by Sanger sequencing.
A unique library of adaptors is used to capture the 16 possible dinucleotide signatures. The library comprises 16 double-stranded DNA adaptors generated from 17 individual oligonucleotides (sequences available in table 2). It contains one constant oligonucleotide (named OB1), which contains a sequence at the 3′ end (5′-gaattcgagctcggtacccg-3′) (SEQ ID NO: 85) for the detection of the ligated products, and 16 individual oligonucleotides, which are composed of a sequence complementary to the constant oligonucleotide and one of the 16 different dinucleotides at their 3′ end (named OB2-OB17).
For detection by DTECTv3-LAMP, the adaptors are prepared from oligonucleotides containing the complementary sequences of the oligo pool to mediate loop amplification. We used two different oligo pools which are used to either detect SARS-CoV-2 ORF1a or geneN (oligo sequences are available in Table 2, Parts 1 and 2).
indicates data missing or illegible when filed
Each oligonucleotide is resuspended at a concentration of 100 μM in TE (10 mM Tris and 0.5 mM EDTA). The annealing reactions are composed of 2.5 μl of the constant oligonucleotide, 2.5 μl of each unique dinucleotide oligonucleotide, and 1× ligase buffer. The reactions are incubated for 5 min at 95° C. to remove any potential secondary structures, followed by a gradual temperature decrease from 95° C. to 15° C. at a ramp rate of 1° C./s. Then, 100 μl H2O is added to dilute the adaptors at 5 μM. Adaptors are stored at −20° C. or −80° C.
The Acu1-tagging PCR utilizes a pair of primers named “Acu1-tagging primer” and “reverse primer” also referred to as “reverse Acu1 proimer”. The objective of the Acu1-tagging PCR is to insert an Acu1 motif 14 bp (5′-CTGAAG-3′) upstream from a targeted dinucleotide, introduce a handle that is used for the detection, and amplify the locus of interest. The Acu1-tagging primer is a 60 nt long oligonucleotide that contains an Acu1 motif as a hairpin 14 np from the 3′ end of the primer. In addition, it also contains a non-complementary handle sequence of 25 nt (5′-GCAATTCCTCACGAGACCCGTCCTG-3′) (SEQ ID NO: 55) that is used for the detection. Therefore, the Acu1 tagging primer has the following architecture: 5′-N(15)CTGAAGN(14)-3′ (SEQ ID NO: 56) with “N” corresponding to A, T, G, or C bases complementary to the targeted locus.
The reverse primer is designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) with a length of “min=25, Opt=27, Max=30” and a Tm of “min=57.0° C., opt=60.0° C., max=63.0° C.”
For DTECTv3-LAMP Acu1-tagging PCR utilizes a different Acu1-tagged primer with the F3 and F2 sequences which are used to detect the SARS-CoV-2 ORF1a or geneN by LAMP. Acu1-tagging primers are 75 nt long oligonucleotide that contains an Acu1 motif as a hairpin 14 np from the 3′ end of the primer. In addition, it also contains a non-complementary handle sequence 5′-CTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGA-3′ (SEQ ID NO: 57) for ORF1a detection and 5′-TGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGG-3′ (SEQ ID NOL: 58) for geneN detection.
The Acu1-tagging PCR is performed in a 25 μl with 1 unit Q5 polymerase (NEB), 1× Q5 buffer, 1 μM of each primer, 10 ng plasmid template, 0.1 mM dNTP in a thermocycler: 95° C. for 30 s; 40 cycles of 95° C. for 10 s, 58° C. for 10 s, 72° C. for 45 s and a final amplification at 72° C. for 1 min. The PCR reaction is loaded on a 2% agarose gel in TAE buffer, and the amplicon is extracted from the gel and column purified (Zymo Research #D4008). The purified Acu1-tagged amplicon is quantified with the nanodrop 2000 and stored at −20° C.
The original DTECT protocol has been conducted as detailed previously. Briefly, DTECT relies on the amplification of the genomic locus of interest using an Acu1-tagging primer. The purified Acu1 tagged amplicon is digested by Acu1 in a 20 μl reaction as follows: 0.2 pmol Acu1 tagged amplicon, 1.25 units Acu1 (NEB #0641) in 1× CutSmart buffer. The digestion is incubated at 37° C. for 1 hour, followed by heat inactivation at 65° C. for 20 min. The SPRI bead (Agencourt AMPure XP magnetic beads) step separates the digested fragments by mixing beads at a DNA:beads ratio of 1:1.8. 10 μl of digestion is mixed with 18 μl of beads by pipetting up and down ten times and incubated at room temperature for 5 min. The tube is then placed on a magnetic rack, and the supernatant is recovered and diluted in 40 μl H2O. Next, the ligation of the adaptors is performed in the following reaction: 6.5 μl H2O, 2 μl of 5× ligase buffer, 0.5 μl T4 ligase (ThermoFisher Scientific), 0.5 μl adaptor, and 0.5 μl of the purified digested product. The ligation reaction is incubated for 1 hour at 25° C. in a thermocycler. The reaction was then stopped by incubating the reaction at 65° C. for 10 min to denature the ligase. The captured material was detected either using quantitative PCR or analytical PCR.
The original DTECT protocol has been conducted as detailed previously. Briefly, DTECT relies on the amplification of the genomic locus of interest using an Acu1-tagging primer. The purified Acu1 tagged amplicon is digested by Acu1 in a 20 μl reaction as follows: 0.2 pmol Acu1 tagged amplicon, 1.25 units Acu1 (NEB #0641) in 1× CutSmart buffer. The digestion is incubated at 37° C. for 1 hour, followed by heat inactivation at 65° C. for 20 min. The SPRI bead (Agencourt AMPure XP magnetic beads) step separates the digested fragments by mixing beads at a DNA:beads ratio of 1:1.8. 10 μl of digestion is mixed with 18 μl of beads by pipetting up and down ten times and incubated at room temperature for 5 min. The tube is then placed on a magnetic rack, and the supernatant is recovered and diluted in 40 μl H2O. Next, the ligation of the adaptors is performed in the following reaction: 6.5 μl H2O, 2 μl of 5× ligase buffer, 0.5 μl T4 ligase (ThermoFisher Scientific), 0.5 μl adaptor, and 0.5 μl of the purified digested product. The ligation reaction is incubated for 1 hour at 25° C. in a thermocycler. The reaction was then stopped by incubating the reaction at 65° C. for 10 min to denature the ligase. The captured material was detected either using quantitative PCR or analytical PCR.
The original DTECT protocol has been conducted as detailed previously. Briefly, DTECT relies on the amplification of the genomic locus of interest using an Acu1-tagging primer. The purified Acu1 tagged amplicon is digested by Acu1 in a 20 μl reaction as follows: 0.2 pmol Acu1 tagged amplicon, 1.25 units Acu1 (NEB #0641) in 1× CutSmart buffer. The digestion is incubated at 37° C. for 1 hour, followed by heat inactivation at 65° C. for 20 min. The SPRI bead (Agencourt AMPure XP magnetic beads) step separates the digested fragments by mixing beads at a DNA:beads ratio of 1:1.8. 10 μl of digestion is mixed with 18 μl of beads by pipetting up and down ten times and incubated at room temperature for 5 min. The tube is then placed on a magnetic rack, and the supernatant is recovered and diluted in 40 μl H2O. Next, the ligation of the adaptors is performed in the following reaction: 6.5 μl H2O, 2 μl of 5× ligase buffer, 0.5 μl T4 ligase (ThermoFisher Scientific), 0.5 μl adaptor, and 0.5 μl of the purified digested product. The ligation reaction is incubated for 1 hour at 25° C. in a thermocycler. The reaction was then stopped by incubating the reaction at 65° C. for 10 min to denature the ligase. The captured material was detected either using quantitative PCR or analytical PCR.
The experiment without bead isolation was carried out following the DTECTv1 procedure, but the bead step was omitted. Without the beads step, the digestion reaction was diluted by adding 100 μl of H2O. This dilution was subsequently used in regular ligation. In addition, enzymes have been diluted in their working buffer; for example, Acu1 was diluted in 1× Cutsmart buffer, and T4 ligase was diluted in 1× ligase buffer. All reactions were conducted in independent duplicates. All incubations were conducted in a thermocycler at the indicated temperature and duration for better reproducibility.
The DTECTv2 protocol relies on DTECTv1 but includes several optimizations. For example, the duration of the digestion/inactivation has been shortened, a dilution in H2O has replaced the bead isolation step, and the adaptor ligation step has been shortened.
The Acu1-tagging PCRs are conducted as described above. The Acu1 digestion/inactivation is performed in 20 μl by mixing 0.2 pmol of Acu1-tagged amplicon with 1.25 units Acu1 in 1× Cutsmart buffer. The digestion is incubated in a thermocycler at 37° C. for 1 min, followed by 1 min at 65° C. The digested reaction is then diluted by the addition of 100 μl H2O and used directly for the ligation. The adaptor ligation is conducted in 10 μl by mixing 2 μl of ligase buffer, 0.5 μl T4 ligase, 0.5 μl of the selected adaptor, and 0.5 μl diluted digestion. The reaction is incubated for 10 min at 25° C. The reaction is stopped by incubating 10 min at 65° C. Finally, analytical or quantitative PCR is performed as detailed above.
The competitor consists of two complementary oligonucleotides, which are annealed to create a double-stranded DNA. The competitor sequences are OB196 5′-AGCCTGTGGTTCCTGAAGATCGCGTCCGAT-3′ (SEQ ID NO: 59) with 5′-CTGAAG-3′ the Acu1 motif, and OB197 5′-ATCGGACGCGATCTTCAGGAACCACAGGCT-3′ (SEQ ID NO: 60) with 5′-CTTCAG-3′ the complementary Acu1 motif. Unlike the Acu1 competitor, the control competitor does not contain an Acu1 motif. The sequences of the two oligonucleotides to make the control competitor are 5′-AGCCTGTGGTTCAAAGTCATCGCGTCCGAT-3′ (SEQ ID NO: 61) and 5′-ATCGGACGCGATGACTTTGAACCACAGGCT-3′ (SEQ ID NO: 62).
To produce the competitors, each oligonucleotide is resuspended at a concentration of 100 μM in TE (10 mM Tris and 0.5 mM EDTA). The annealing reactions are composed of 2.5 μl of each complementary oligonucleotide and 1× ligase buffer. The reactions are incubated for 5 min at 95° C. to remove any potential secondary structures, followed by a gradual temperature decrease from 95° C. to 15° C. at a ramp rate of 1° C./s. Then, the competitor is diluted at 5 μM. Competitors are stored at −20° C.
The one-pot DTECTv3 protocol merges DNA ligation and Acu1 digestion in a single tube. It utilizes an optimized quantity of Acu1-tagged amplicon compatible with the one-pot digestion-ligation reaction. The Acu1-tagging PCRs are conducted as described above. The reactions are conducted in a single tube but separated in two independent steps as follows: 0.005 pmol of Acu1 tagged amplicon is digested in a 7 μl reaction by mixing 1 μl Cutsmart buffer, 1.25 μl of diluted Acu1 (Acu1 was diluted 1/10th in 1× Cutsmart buffer) and completed with H2O. The digestion is incubated for 1 min at 37° C. and 1 min at 65° C. in a thermocycler. Then, 2 μl ligase buffer, 0.5 μl of the selected adaptor, and 0.5 μl T4 ligase were added to the reaction and incubated for 10 min at 25° C. The ligation was stopped by incubation at 65° C. for 10 min. Finally, analytical or quantitative PCR is performed as detailed above.
DTECTv3 only requires an Acu1-tagged amplicon. A 2× DTECTv3 master mix is prepared as follows (recipe to prepare 400 DTECTv3 reactions): 290 μl H2O, 400 μl 5× ligase buffer, 200 μl competitor (1 pmol/μl), 10 μl Acu1 (10 u/μl) and 100 μl T4 ligase (1 u/μl). The capture is conducted in a 5 μl reaction as follows: 2.5 μl 2× DTECTv3 master mix, 0.25 μl adaptor, and 0.005 pmol Acu1 tagged amplicon. The digestion is incubated in a thermocycler at 25° C. for 1 min, 10 min or 1 hour. The reaction is then stopped by incubating the reaction at 65° C. for 30 s. The captured material is then detected either using quantitative PCR, analytical PCR or DTECT-LAMP.
For detection of the captured product by quantitative PCR, a qPCR master mix is prepared. The recipe to prepare 100 DTECTv3-qPCR reactions (900 μl total) is as follows: 500 μl of 2× SYBR Green master mix, 380 μl H2O, 10 μl of primer OB1 (100 μM), and 10 μl of primer OB2 (100 μM). 9 μl of qPCR master mix is added in each qPCR well and 1 μl of DTECTv3 is added.
An oligo pool containing LAMP oligos F3, FIP, B3, BIP and LB is prepared. The recipe to prepare 100 μl of oligo pool master mix for LAMP detection is as follows: 20 μl H2O, 4 μl F3 (100 μM), 32 μl FIP (100 μM), 4 μl B3 (100 μM), 32 μl BIP (100 μM), 8 μl LB (100 μM). Sequences of oligonucleotides are in Table 1.
The LAMP detection reaction is prepared as follows: 5 μl 2× WarmStart colorimetric LAMP (NEB #M1800), 0.4 μl H2O, 1.6 μl betaine (5 M), 0.5 μl oligo pool, and 1 μl of DTECTv3 capture (diluted 1/1000th in H2O), added in a WarmStart colorimetric LAMP 2× Master mix (NEB #M1800) in a 10 μl reaction and incubated at 65° C. until the red change turned yellow.
To quantify the LAMP reaction, Spectra Max iD3 was used to measure the absorbance levels at wavelengths 415 and 560 nm by incubating the reaction for 2 hours at 65° C.
A standard curve to determine the efficiency of the qPCR amplification and the linearity of the amplification was generated with a plasmid that contains a DTECT ligation product (Addgene #139333) using primers OB18 and OB19 (sequences in Table 1). The linearity of the standard curve has the mathematical formula: y=−3.3245×+7.5504.
Each sample analyzed by qPCR is tested in technical duplicates, and the mean Ct for each sample is calculated. The capture score is defined as the concentration of the captured material for each sample multiplied by 10{circumflex over ( )}6. It is measured as follow: Capture score=(10{circumflex over ( )}[(Mean Ct−7.5504)/−3.3245])×10{circumflex over ( )}6. The reported capture score corresponds to the mean of two independent experiments and is shown as LOG10.
The efficiency score corresponds to the absolute value of the difference in Ct between the specific and non-specific adaptors. It was calculated using the formula: “=ABS(Ct specific adaptor−Ct non-specific adaptor)”. Thus, the reported efficiency score corresponds to the mean of two independent experiments.
The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
This application claims priority to United States Provisional Patent Application U.S. 63/209,619, filed on 11 Jun. 2021, the entire contents of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050914 | 6/8/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63209619 | Jun 2021 | US |
