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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• PBNJ specific binding to the reference sequence is competitive to the labeled probe binding to the reference sequence and suppresses labeled probe bound to the reference sequence relative to amplification; thereby discriminating the target sequence from the reference sequence.
• the competitive concentration of PBNJ:probe will depend on the application of interest and, therefore, is compatible with a range of concentrations. Examples include, but are not limited to ratios of between 0.2 and 20.
• PCR is dPCR and the dPCR comprises partition or droplet-based PCR and the PBNJ reduces or eliminates signal associated with a lower efficiency, non-specific off-target amplification, thereby increasing a signal to noise ratio for specific amplification of the target sequence.
• FIGS. 4A-4C show amplification results as detected by fluorescence signal, in the presence of the PBNJ, including multiplexed results.
• FIG. 4A shows amplification for two probes in the same channel.
• FIG. 4B shows amplification for two probes in the same, alternative channel.
• FIG. 4C shows amplitude tuning for multiplex strategies using several channels and 2-Dimension based analysis.
• FIGS. 5A-5D show that concentrations of PBNJ relative to dual -labeled probe to the 484K mutation of SARS CoV-2 reduce non-specific signal at concentration ratios of PBNJ:probe between 2: 1 and 8: 1.
• [PBNJ] 400 nM.
• [PBNJ] 800 nM.
• [PBNJ] 1600 nM.
• FIG. 6 shows titration of a PBNJ to reduce fluorescence levels of off-target amplification using SARS-CoV-2 variant mutation assays allowing improved discrimination of the 417N mutation.
• FIGs 12A-12B illustrate impacts of PBNJ:probe ratio, length, and presence of
• a blood sample can be fractionated into serum, plasma or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes).
• a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample.
• biological sample also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example.
• biological sample also includes materials derived from a tissue culture or a cell culture.
• a “biological sample” obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual.
• the biological sample is used to test for mutations associated with an elevated risk of disease.
• the reference sequence is reflective of a low-disease condition state and the target sequence has one or more nucleotide changes in the reference sequence reflective of an elevated disease condition risk or presence of disease.
• the biological sample is used to test for mutations associated with an elevated risk of cancer, dementia and/or cardiovascular conditions.
• the biological sample is used to test for a variant of a pathogen, including a pathogen that is a virus.
• the reference sequence when testing for a variant of a virus, is from a wild-type virus or a parent virus and the target sequence comprises at least one mutation in the reference sequence.
• the biological sample is from wastewater, environmental sample, bodily fluid, tissue, cell culture, or tumor.
• discriminating refers to making a distinction or distinguishing between two or more things. In certain aspects, “discriminating” refers to distinguishing between a reference oligonucleotide sequence and a target oligonucleotide sequence.
• non-specifically binds refers to binding or hybridization of a binding agent which is not correlated with the specificity of the binding agent.
• the binding agent is an oligonucleotide which non-specifically hybridizes to an oligonucleotide sequence which is not completely complementary to the sequence of the oligonucleotide binding agent.
• target or “target sequence” are used interchangeably to refer to a nucleic acid that hybridizes to a primer and can be detected and quantified by dPCR and qPCR analysis.
• Target or target sequence is used broadly to refer to any oligonucleotide sequence of interest, including a sequence associated with a pathogen (e.g., virus or bacteria) and a sequence associated with a patient genome (e.g., a mammal, such as a human).
• a labeled probe binds specifically to the target or target sequence and, therefore, has complementarity to the target or target sequence.
• a primer does not need to reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
• a primer may further comprise a "tail" comprising additional nucleotides at the 5' end of the primer that are non- complementary to the template.
• the lengths of primers range between 7-100 nucleotides in length, such as 10-30, 15-60, 20-40, and so on, more typically in the range of between 15-35 nucleotides in length, and any sub-ranges thereof. Shorter primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
• the term "primer site” or “primer binding site” refers to the segment of the target DNA to which a primer hybridizes.
• sequence complementarity refers to the standard arrangement of bases in nucleotides in relation to their opposite pairing, such as thymine being paired with adenine and cytosine being paired with guanine. In certain aspects, sequence complementarity is complete or exact complementarity at all base positions within an oligonucleotide. RNA has uracil instead of thymine.
• the PBNJ has a C3 -spacer modification at the 3 ’-end to prevent 5’ to 3’ elongation by a DNA polymerase.
• the PBNJ and the probe compete during hybridization, increasing target specificity of the probe due to change in affinity for respective targets as shown in FIG. 1, particularly for PBNJ in excess.
• the modification at the 3’-end of the PBNJ is a C3-spacer or a 3’ quencher such as a black hole quencher.
• the PBNJ can also be modified with extended carbon-spacers (C6, C9, C12, et).
• the ratio of probe to PBNJ is such that the concentration of PBNJ is in excess of the concentration of the probe. In certain aspects, a ratio of PBNJ concentration to dual-labeled probe concentration is at least 1.5: 1. In certain further aspects, the PBNJ acts as a PCR blocker during amplification cycles, leading to mutant allele enrichment. In certain aspects, reactions can be multiplexed to utilize two or more PBNJs.
• FIGs 2A-4C One practical impact of the methods, kits, and compositions provided herein is illustrated in FIGs 2A-4C.
• use of PBNJ effectively reduces detection of undesired amplicons show amplification results as detected by fluorescence signal (compare FIG. 2A without PBNJ and FIG. 2B with PBNJ).
• Possibly interfering signal associated with non-specific amplification is reduced by use of the PBNJ provided therein. This improves the ability to properly set a threshold 200.
• FIG. 2A shows specific amplification and nonspecific amplification results, wherein the probe binds both on-target and off-target molecules in the reaction.
• FIG. 2B illustrates improved detection of specific amplification, wherein there is an extinction of the non-specific signal with PBNJ.
• the probe output amplitude is tuned by providing the PBNJ at a lower concentration.
• a plurality of probe output amplitudes is tuned for multiplex detection of a plurality of target sequences in a single or a multichannel fluorescence detector.
• Kits are disclosed for practicing the methods disclosed herein. Kits provide for discriminating a target sequence from a reference sequence in a biological sample by dPCR or RT-PCR.
• kits comprise buffers, primers, polymerase, one or more labeled probes and one or more PBNJ’s.
• a forward primer is provided at a concentration of between 50 nM and 1100 nM.
• a reverse primer is provided at a concentration of between 50 nM and 1100 nM.
• labeled- probe is provided at a concentration of 20-800 nM.
• one or more PBNJ is provided at a concentration that is between 0.25x and 16x the concentration of the labeled- probe.
• This example describes the use of PBNJs to reduce off target amplification using E484K probe.
• Dual-labeled probe was designed towards the mutant 484K sequence.
• concentrations of reagents utilized were as follows: common forward primer - 500 nM; common reverse primer - 500nM; dual-labeled probe to 484K - 200 nM; and PBNJ concentration was variable.
• Assays were conducted on the QIAGEN - QIAcuity® instrument.
• FIG. 5B-5D show results for increasing concentrations of PBNJ.
• FIG. 5B shows that off-target E484 signal is extinct when PBNJ is added at a competitive concentration of 400nM, which corresponds to a ratio of [2: 1] (PBNJ:Probe).
• excess PBNJ up to ratio 8: 1 does not affect on-target detection, while extinguishing non-specific signal.
• This example describes the use of PBNJs to reduce off target amplification using 417N probe.
• Dual-labeled probe was designed towards the mutant 417N sequence.
• the dual labeled probe was used on a mixture of template consisting of Parental, Beta, Gamma, and Delta variants of SARS-CoV-2.
• the probe produced a high amplitude, population of droplets resulting from on-target amplification on template from the beta variant of SARS-CoV-2 which contains the 417N mutation (highlighted by arrow in FIG. 6) and off-target, lower amplitude populations of partitions due to off-target, less efficient amplification.
• Assays were conducted on the QIAGEN - QIAcuity® instrument.
• KRAS G12C probe Dual-labeled probe was designed towards the mutant KRAS G12C sequence.
• concentrations of reagents utilized were as follows: common forward primer - 900 nM; common reverse primer - 900nM; dual-labeled probe to G12C - 250 nM; and PBNJ -500 nM.
• Assays were conducted on the BIORAD - DROPLET DIGITAL QX200 instrument. See Tables 2-5 for sequence information and concentrations tested.
• the probe produced a high amplitude population of droplets on G12C mutation containing template. This probe also produced lower amplitude off-target droplet populations on WT template and G12R template as demonstrated by the presence of lower amplitude, off target populations in wild-type template (B01, E01) and in alternative G12R mutation containing template (E01), as shown in FIG. 7.
• FIGs. 13A-13B confirms that PBNJs do not adversely impact target amplification on pure KRAS-G 12C (FIG. 13A) or EGFR-T790M (FIG. 13B) synthetic DNA, for either 80% or 100% PBNJ length (relative to probe).
• the bottom panels illustrate that any of a PBNJ concentration (ranging from Ox to 6x) does not result in a significantly different concentration of quantified amplicon.
• PBNJs are synthesized with either 3’ C3 or BHQ-1 polymerase extension blockers and tested against on- and off-target templates in RT-PCR with TaqPath® (Thermo) on the Bio-Rad CFX-96. As seen in FIGs. 14A-14C, E484 PBNJs are able to efficiently block nonspecific amplification without any loss of on-target detection.
• PBNJs can be modified with either C3 (and longer chains, e.g., C6 and the like) as well as BHQ-1 and show efficient nonspecific amplification blocking. Furthermore, PBNJs are compatible with RT-PCR.

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• 2022
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