WO2020105058A1 - Procédés de mesure de petites différences d'abondance d'acides nucléiques - Google Patents

Procédés de mesure de petites différences d'abondance d'acides nucléiques

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WO2020105058A1
WO2020105058A1 PCT/IL2019/051286 IL2019051286W WO2020105058A1 WO 2020105058 A1 WO2020105058 A1 WO 2020105058A1 IL 2019051286 W IL2019051286 W IL 2019051286W WO 2020105058 A1 WO2020105058 A1 WO 2020105058A1
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complementarity
sample
nucleic acid
acid molecule
primers
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PCT/IL2019/051286
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Chen AZULAY
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Individual
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention is in the field of nucleic acid detection and quantification.
  • PCR is a well-established method for amplifying specific sections from template DNA.
  • Real-time PCR or quantitative PCR is a further development of conventional PCR, in which a fluorescent dye, that increase its emission according to the amount of the amplified DNA produced, is added in addition to the basic PCR reagents (template DNA, primers, enzymes, probes nucleotides and a buffer solution). The dye emission can then be detected by a spectrum specific detector. The data is transmitted to a computer for the calculation of the reaction plot, and the plot presents the change in emission of the specific spectrum for each PCR cycle.
  • a reaction has to reach a specific threshold in order to be recorded as reaction and not just fluorescent artifacts (“background noise”). Once the cycle that reaches this threshold (Ct) is found, Ct values from different reactions can be compared to quantify relative differences in amounts of the template DNA present in the tested samples.
  • background noise background noise
  • PCR The ability of PCR to be truly quantitative, however, is limited by a number of factors.
  • the efficiency of the primers used is paramount, in that a 100% efficient primer will show a reaction with exactly double the amount of template will reach the threshold exactly one cycle earlier.
  • Primers are designed to have 100% complementarity to the target template sequence and to have as little complementarity to any other sequences as possible. If a primer binds to off-targets due to partial complementarity to these targets, the efficiency of the primer will be decreased.
  • duplex qPCR there exists a method for the detection and quantification of two genes at once called duplex qPCR.
  • two genes are amplified in one reaction tube, where each gene has its own set of primers and probes.
  • the two probes have different fluorescence and thus the two amplification products can be measured distinctly. It is much harder to create a stable primer-target duplex with 100% efficiency in duplex reactions as compared to single gene reactions due to interaction between the different reagents in the tube.
  • cfDNA Cell free DNA
  • cffDNA a DNA fragments that can be found in the blood
  • cffDNA a fetal origin
  • the origin of the cfDNA cannot be readily discerned and the fetal fraction is estimated to be only 3-10% of the total cfDNA. Therefore, changes in the cffDNA represent only a very small fraction of the overall cfDNA that can be extracted from the mother.
  • cfDNA based fetal monitoring assays have been achieved, but these methods rely on next generation sequencing of the entire cfDNA sample. This is expensive, time consuming, wasteful and also often produces inaccurate results.
  • a PCR -based method of cfDNA fetal monitoring would greatly simplify and cheapen the process, making more accessible and potentially more accurate.
  • the present invention provides methods and kits for detecting and measuring small differences in amounts of a target nucleic acid sequence in a test sample as compared to a control sample by using competitive duplex qPCR.
  • a method for detecting or measuring a small difference in an amount of a first nucleic acid molecule in a first sample as compared to a second sample comprising:
  • qPCR duplex quantitative polymerase chain reaction
  • API amplification product of the first nucleic acid molecule
  • AP2 amplification product of the second nucleic acid molecule
  • a method for detecting or measuring a small difference in an amount of a first nucleic acid molecule in a first sample as compared to a second sample comprising:
  • qPCR duplex quantitative polymerase chain reaction
  • a first set of primers with at least 90% complementarity to the first nucleic acid molecule, at least 10% complementarity to a second nucleic acid molecule and greater complementarity to the first nucleic acid molecule than to the second nucleic acid molecule; and ii. a second set of primers with at least 10% complementarity to the first nucleic acid molecule, at least 90% complementarity to the second nucleic acid molecule and greater complementarity to the second nucleic acid molecule than to the first nucleic acid molecule; and
  • API amplification product of the first nucleic acid molecule
  • AP2 amplification product of the second nucleic acid molecule
  • the method further comprises performing duplex qPCR with the first and second set of primers on nucleic acids from the second sample and measuring API and AP2 in the second sample.
  • the second sample is a control sample and the ratio of API to AP2 in the control sample is known.
  • the 10-90% complementarity to the second nucleic acid molecule is 10-45% and wherein the 10-90% complementarity to the first nucleic acid molecule is 10-45%.
  • the at least 10% complementarity to the second nucleic acid molecule is 10-45% complementarity and the at least 10% complementarity to the first nucleic acid molecule is 10-45% complementarity.
  • the nucleic acids from the first sample are selected from the group consisting of:
  • cfDNA cell-free DNA
  • fetal DNA from the sample
  • cffDNA fetal cfDNA
  • the first nucleic acid is N-[016] According to some embodiments, the first nucleic acid
  • a. comprises a disease-associated mutation
  • b. is an oncogene
  • c. is a marker for a genetic disorder
  • d. comprises a marker for a genetic disorder
  • e. is from a pathogen
  • the marker for a genetic disorder is a trisomy of a chromosome.
  • the genetic disorder is Down syndrome and the marker is trisomy of chromosome 21.
  • the pathogen is selected from a virus, a bacterium and a fungus.
  • the small difference in an amount is less than a 10% change in the amount.
  • the small difference in an amount is between a 5% and a 0.2% change in the amount.
  • the second nucleic acid molecule is a control molecule, and wherein the abundance of the control molecule is the same in the first and second samples.
  • the qPCR is a multiplex PCR and further comprises at least one additional set of primers with at least 90% complementarity to a target nucleic acid molecule and 10-90% complementarity to a control nucleic acid molecule, and wherein no nucleic acid molecule has greater than 10% complementarity to more than two sets of primers.
  • the qPCR is a multiplex PCR and further comprises at least one additional set of primers with at least 90% complementarity to a target nucleic acid molecule, at least 10% complementarity to a control nucleic acid molecule, and greater complementarity to the target nucleic acid molecule than to the control nucleic acid molecule, and wherein no nucleic acid molecule has greater than 10% complementarity to more than two sets of primers.
  • the qPCR is Taqman qPCR.
  • the qPCR comprises a first probe and a second probe and wherein the first probe comprises 90-100% complementarity to the API and 10-90% complementarity to the AP2, and the second probe comprises 90-100% complementarity to the AP2 and 10-90% complementarity to the API.
  • the qPCR comprises a first probe and a second probe and wherein the first probe comprises at least 90% complementarity to the API, at least 10% complementarity to the AP2 and greater complementarity to the API than the AP2, and the second probe comprises at least 90% complementarity to the AP2, at least 10% complementarity to the API and greater complementarity to the AP2 than the API.
  • the 10-90% complementarity to the AP2 is 10-45%
  • the 10-90% complementarity to the API is 10-45%
  • At least 10% complementarity to the AP2 is 10-45% complementarity and at least 10% complementarity to the API is 10-45% complementarity.
  • At least one reagent of the qPCR reaction is present in a low amount, wherein the low amount is an amount below an amount recommended by a manufacturer of the reagent.
  • the low amount of at least one reagent is selected from:
  • At least one cycle of the qPCR has a reduced extension time as compared to the extension time recommended by a manufacturer, a reduced annealing time as compared to the annealing time recommended by a manufacturer, or both.
  • the measuring an amplification product allows for determining a cycle at which the amount of the product reaches a predetermined threshold (Ct), and the method further comprises calculating the change in the Ct (ACt) between the first sample and the second sample for the first nucleic acid molecule and the second nucleic acid molecule.
  • Ct predetermined threshold
  • the method further comprises subtracting the ACt for the first nucleic acid molecule from the ACt for the second nucleic acid molecule to receive a total change in the amount of the first nucleic acid molecule.
  • the first sample is a sample to be tested and the second sample is a control sample.
  • the sample to be tested is selected from a group consisting of:
  • c. is at risk of being infected with a pathogen
  • the pathogen is selected from a virus, a bacterium and a fungus.
  • the pregnant subject carries a fetus at risk of carrying a genetic disorder.
  • kits comprising two sets of primers, wherein the first set of primers comprises at least 90% complementarity to a target sequence of interest, and 10-90% complementarity to a control sequence, and wherein the second set of primers comprises at least 90% complementarity to the control sequence and 10-90% complementarity to the target sequence.
  • kits comprising two sets of primers, wherein the first set of primers comprises at least 90% complementarity to a target sequence of interest, at least 10% complementarity to a control sequence, and greater complementarity to the target sequence of interest than to the control sequence and wherein the second set of primers comprises at least 90% complementarity to the control sequence, at least 10% complementarity to the target sequence of interest and greater complementarity to the control sequence than to the target sequence of interest.
  • the 10-90% complementarity to a control sequence is 10- 45% and the 10-90% complementarity to the target sequence is 10-45%.
  • At least 10% complementarity to a control sequence is 10-45% complementarity and at least 10% complementarity to the target sequence is 10-45% complementarity.
  • the kit further comprises at least one reagent for qPCR.
  • the at least one reagent is a first probe and a second probe, and wherein the first probe comprises 90-100% complementarity to an amplification product of the first set of primers (API) and 10-90% complementarity to an amplification product of the second set of primers (AP2), and the second probe comprises 90-100% complementarity to the AP2 and 10-90% complementarity to the API.
  • the first probe comprises 90-100% complementarity to an amplification product of the first set of primers (API) and 10-90% complementarity to an amplification product of the second set of primers (AP2)
  • the second probe comprises 90-100% complementarity to the AP2 and 10-90% complementarity to the API.
  • the at least one reagent is a first probe and a second probe
  • the first probe comprises at least 90% complementarity to an amplification product of the first set of primers (API), at least 10% complementarity to an amplification product of the second set of primers (AP2), and greater complementarity to the API than to the AP2
  • the second probe comprises at least 90% complementarity to the AP2, at least 10% complementarity to the API and greater complementarity to the AP2 than to the API.
  • the 10-90% complementarity to AP2 is 10-45%
  • the 10-90% complementarity to the API is 10-45%
  • kits of the invention for use in detecting or measuring small differences in an amount of the target sequence of interest in a test sample as compared to a control sample.
  • the kit further comprises a table of values of PCR results in at least one control sample.
  • the small difference in an amount is selected from: less than a 10% change in the amount and between a 5% and a 0.2% change in the amount.
  • the target sequence of interest is at least one of:
  • cfDNA cell-free DNA
  • fetal cfDNA fetal cfDNA
  • h. comprises a disease-associated mutation
  • i. is an oncogene
  • j is a marker for a genetic disorder
  • k. comprises a marker for a genetic disorder
  • l. is from a pathogen
  • Figures 1A-B Amplification plots showing the abundance of the (1A) Y chromosome and (IB) X chromosome in 8 technical replicates from a control sample and a test sample.
  • Figure 2 A bar graph of relative abundance of the Y and X chromosomes as calculated from the samples in Figure 1A and IB.
  • Figure 3 A bar graph of relative abundance of the Y and X chromosomes from 2 controls and 3 test samples with varying amounts of X chromosome.
  • Figure 4 A best fit curve of relative X abundance and quantity distance as derived from the data presented in Figures 2 and 3.
  • Figure 5 A bar graph of relative abundance of chromosome 21 and X chromosomes from cfDNA from pregnant mothers carrying a female fetus, a male fetus and boy /girl twins.
  • Figure 6 A bar graph of relative abundance of chromosome 21 and X chromosomes from cfDNA from mothers with healthy (Samples 2-4) and Down syndrome (Sample 1) fetuses.
  • the present invention provides, in some embodiments, methods and kits for detecting and measuring small differences in amounts of a target nucleic acid sequence in a test sample as compared to a control sample.
  • the present invention is based, in part, on the surprising finding that competitive qPCR reactions wherein the primers used to amplify one target have a partial complementarity to a second target can enhance the resolution of the PCR and allow for detection and quantification of very small changes in abundance of the target template, even changes as small as an increase of 0.2% of the starting material.
  • a method for detecting or measuring a small difference in an amount of a first nucleic acid molecule in a first sample as compared to a second sample comprising:
  • qPCR duplex quantitative polymerase chain reaction
  • API amplification product of the first nucleic acid molecule
  • AP2 amplification product of the second nucleic acid molecule
  • a method for detecting or measuring a small difference in an amount of a first nucleic acid molecule in a first sample as compared to a second sample comprising:
  • qPCR duplex quantitative polymerase chain reaction
  • a first set of primers with at least 70% complementarity to the first nucleic acid molecule, at least 10% complementarity to a second nucleic acid molecule and greater complementarity to the first nucleic acid molecule than to the second nucleic acid molecule;
  • a second set of primers with at least 10% complementarity to the first nucleic acid molecule, at least 70% complementarity to the second nucleic acid molecule and greater complementarity to the second nucleic acid molecule than to the first nucleic acid molecule;
  • API amplification product of the first nucleic acid molecule
  • AP2 amplification product of the second nucleic acid molecule
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine "G,” a thymine “T” or a cytosine "C”) or RNA (e.g., an A, a G, an uracil "U” or a C).
  • nucleic acid molecule includes but not limited to single- stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double- stranded DNA (dsDNA), small RNAs, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides.
  • ssRNA single- stranded RNA
  • dsRNA double-stranded RNA
  • ssDNA single-stranded DNA
  • dsDNA double- stranded DNA
  • small RNAs circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides.
  • isolated nucleic acid molecule refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature.
  • a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • the isolated DNA is cDNA.
  • the isolated nucleic acid is any one of DNA, RNA, and cDNA.
  • the term“primer” includes an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Primers within the scope of the present invention bind adjacent to a target sequence.
  • A“primer” may be considered a short polynucleotide, generally with a free 3'- OH group that binds to a target or template potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
  • Primers of the invention are comprised of nucleotides ranging from 8 to 30 nucleotides.
  • the primer is at least 8 nucleotides, or alternatively at least 9 nucleotides, or alternatively at least 10 nucleotides, or alternatively at least 11 nucleotides, or alternatively at least 12 nucleotides, or alternatively at least 13 nucleotides, or alternatively at least 14 nucleotides, or alternatively at least 15 nucleotides, or alternatively at least 16 nucleotides, or alternatively at least 17 nucleotides, or alternatively, at least 18 nucleotides, or alternatively, at least 19 nucleotides, or alternatively, at least 20 nucleotides, or alternatively, at least 21 nucleotides, or alternatively, at least 22 nucleotides, or alternatively, at least 23 nucleotides, or alternatively, at least 24 nucleotides, or alternatively, at least 25 nucleotides, or
  • Complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine.
  • a set of primers is said to have a specific complementarity if each and every primer of the set has that complementarity. That is if a set of two primers is said to have between 90 and 100% complementarity to a sequence, then the first primer of the set will have between 90 and 100% complementarity to the sequence and the second primer of the set will have between 90 and 100% complementarity to the sequence. Similarly, if the first primer has 100% complementarity and the second has 88% complementarity, such as set would not be said to have between 90 and 100% complementarity even though the average of the two primers would be 94% complementary.
  • At least 70% is at least 75, 80, 85, 90, 93, 95, 97, 98, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 70% is 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, 98-100%, or 99- 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 10% is at least 10, 15, 20, 25, 30, 35, 40, 45, or 50%. Each possibility represents a separate embodiment of the invention.
  • At least 10% is 10-99%, 15-99%, 20-99%, 25-99%, 30-99%, 35-99%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 10-90%, 15- 90%, 20-90%, 25-90%, 30-90%, 35-90%, 10-80%, 15-80%, 20-80%, 25-80%, 30-80%, 35- 80%, 10-70%, 15-70%, 20-70%, 25-70%, 30-70%, 35-70%, 10-60%, 15-60%, 20-60%, 25-60%, 30-0%, 35-60%, 10-50%, 15-50%, 20-50%, 25-50%, 30-50%, 35-50% , 10-40%, 15-40%, 20-40%, 25-40%, 30-40%, 35-40%, 10-35%, 15-35%, 20-40%, 25-35%, 30-35%, 10-30%, 15-30%, 20- 30%, or 25-30%.
  • Each possibility represents a separate embodiment of the invention.
  • the first set ofprimers comprises 70-100%, 75-100%, 80-100%, 85- 100%, 90-100%, 95-100%, 97-100%, 98-100%, or 99-100% complementarity to a first nucleic acid molecule.
  • the first set of primers comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementarity to a first nucleic acid molecule.
  • the first set of primers comprises 100% complementarity to a first nucleic acid molecule.
  • the first set of primers comprises at least 99% complementarity to a first nucleic acid molecule. In some embodiments, the first set of primers comprises at least 97% complementarity to a first nucleic acid molecule. In some embodiments, the first set of primers comprises at least 95% complementarity to a first nucleic acid molecule. In some embodiments, the first set of primers comprises at least 92% complementarity to a first nucleic acid molecule. In some embodiments, the first set of primers comprises at least 90% complementarity to a first nucleic acid molecule. In some embodiments, the first set of primers comprises at least 80% complementarity to a first nucleic acid molecule.
  • the first set of primers comprises between 10-99%, 15-99%, 20- 99%, 25-99%, 30-99%, 35-99%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 10-90%, 15-90%, 20-90%, 25-90%, 30-90%, 35-90%, 10-80%, 15-80%, 20-80%, 25-80%, 30-80%, 35- 80%, 10-70%, 15-70%, 20-70%, 25-70%, 30-70%, 35-70%, 10-60%, 15-60%, 20-60%, 25-60%, 30-0%, 35-60%, 10-50%, 15-50%, 20-50%, 25-50%, 30-50%, 35-50% , 10-40%, 15-40%, 20-40%, 25-40%, 30-40%, 35-40%, 10-35%, 15-35%, 20-40%, 25-35%, 30-35%, 10-30%, 15-30%, 20- 30%, or 25-30%, complementarity to a second nucleic acid
  • the first set of primers comprises at least 1,.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% complementarity to a second nucleic acid molecule.
  • the first set of primers comprises between 10-45% complementarity to a second nucleic acid molecule.
  • the first set of primers comprises between 10-50% complementarity to a second nucleic acid molecule.
  • the first set of primers comprises between 20-30% complementarity to a second nucleic acid molecule.
  • the first set of primers comprises between 14-45% complementarity to a second nucleic acid molecule.
  • the first set of primers comprises between 15-45% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises between 10-42% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises between 14-42% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises between 15-25% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises between 10-80% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises between 10-85% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises at least 10% complementarity to a second nucleic acid molecule. In some embodiments, the first set of primers comprises at least 14% complementarity to a second nucleic acid molecule.
  • the second set of primers comprises between 10-99%, 15-99%, 20- 99%, 25-99%, 30-99%, 35-99%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 10-90%, 15-90%, 20-90%, 25-90%, 30-90%, 35-90%, 10-80%, 15-80%, 20-80%, 25-80%, 30-80%, 35- 80%, 10-70%, 15-70%, 20-70%, 25-70%, 30-70%, 35-70%, 10-60%, 15-60%, 20-60%, 25-60%, 30-0%, 35-60%, 10-50%, 15-50%, 20-50%, 25-50%, 30-50%, 35-50% , 10-40%, 15-40%, 20-40%, 25-40%, 30-40%, 35-40%, 10-35%, 15-35%, 20-40%, 25-35%, 30-35%, 10-30%, 15-30%, 20- 30%, or 25-30%, complementarity to the first nucleic acid
  • the second set of primers comprises at least 1,.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% complementarity to the first nucleic acid molecule.
  • the second set of primers comprises between 10-45% complementarity to a first nucleic acid molecule.
  • the second set of primers comprises between 10-50% complementarity to a first nucleic acid molecule.
  • the second set of primers comprises between 20-30% complementarity to a first nucleic acid molecule.
  • the second set of primers comprises between 14-45% complementarity to a first nucleic acid molecule.
  • the second set of primers comprises between 15- 45% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises between 10-42% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises between 14-42% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises between 15- 25% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises between 10-80% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises between 10-85% complementarity to a first nucleic acid molecule. In some embodiments, the second set of primers comprises at least 10% complementarity to the first nucleic acid molecule. In some embodiments, the second set of primers comprises at least 14% complementarity to the first nucleic acid molecule.
  • the second set of primers comprises 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, 98-100%, or 99-100% complementarity to the second nucleic acid molecule.
  • the second set of primers comprises at least 70, 75, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementarity to the second nucleic acid molecule.
  • the second set of primers comprises 100% complementarity to the second nucleic acid molecule.
  • the second set of primers comprises at least 99% complementarity to a second nucleic acid molecule. In some embodiments, the second set of primers comprises at least 97% complementarity to a second nucleic acid molecule. In some embodiments, the second set of primers comprises at least 95% complementarity to a second nucleic acid molecule. In some embodiments, the second set of primers comprises at least 92% complementarity to a second nucleic acid molecule. In some embodiments, the second set of primers comprises at least 90% complementarity to a second nucleic acid molecule. In some embodiments, the second set of primers comprises at least 80% complementarity to a second nucleic acid molecule.
  • the first set of primers and the second set of primers comprise the same level of complementarity to their primary targets (i.e. the target that they bind with greater complementarity). In some embodiments, the first set of primers and the second set of primers comprise the same level of complementarity to their secondary targets (i.e. the target they bind with lower complementarity). In some embodiments, the same level of complementarity is roughly the same level of complementarity. In some embodiments, roughly the same is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% of each other. Each possibility represents a separate embodiment of the invention. In some embodiments, the first set of primers and the second set of primers comprise different levels of complementarity to their primary targets. In some embodiments, the first set of primers and the second set of primers comprise different levels of complementarity to their secondary targets
  • primers will have a greater complementarity for the sequence which they are intended to amplify than for the other sequence in the duplex PCR.
  • the first set of primers has greater complementarity to the first nucleic acid molecule than the second.
  • the second set of primers has greater complementarity to the second nucleic acid molecule than the first.
  • either primer set may have equal complementarity to both sequences.
  • the partial complementarity to a second sequence will be predominantly at the 5’ or 3’ end of the primer.
  • the forward primer will have partial complementarity primarily at the 5’ end of the primer.
  • the forward primer will have partial complementarity primarily at the 3’ end of the primer.
  • the reverse primer will have partial complementarity primarily at the 3’ end of the primer.
  • the reverse primer will have partial complementarity primarily at the 5’ end of the primer.
  • a forward primer of the first primer set will have at least 1, at least 2 or at least 3 complementary bases to the second molecule within the first 5 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a forward primer of the first primer set will have at least 1 , at least 2 or at least 3 complementary bases to the second molecule within the last 5 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a forward primer of the first primer set will have at least 1 , at least 2 or at least 3 complementary bases to the second molecule within the first 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a forward primer of the first primer set will have at least 1, at least 2 or at least 3 complementary bases to the second molecule within the last 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the first primer set will have at least 1 , at least 2 or at least 3 complementary bases to the second molecule within the last 5 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the first primer set will have at least 1 , at least 2 or at least 3 complementary bases to the second molecule within the first 5 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the first primer set will have at least 1, at least 2 or at least 3 complementary bases to the second molecule within the last 10 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a reverse primer of the first primer set will have at least 1, at least 2 or at least 3 complementary bases to the second molecule within the first 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a forward primer of the second primer set will have at least 1, at least 2 or at least 3 complementary bases to the first molecule within the second 5 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a forward primer of the second primer set will have at least 1 , at least 2 or at least 3 complementary bases to the first molecule within the last 5 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a forward primer of the second primer set will have at least 1, at least 2 or at least 3 complementary bases to the first molecule within the second 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a forward primer of the second primer set will have at least 1, at least 2 or at least 3 complementary bases to the first molecule within the last 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the second primer set will have at least 1 , at least 2 or at least 3 complementary bases to the first molecule within the last 5 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the second primer set will have at least 1 , at least 2 or at least 3 complementary bases to the first molecule within the second 5 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • a reverse primer of the second primer set will have at least 1, at least 2 or at least 3 complementary bases to the first molecule within the last 10 bases of the primer. Each possibility represents a separate embodiment of the invention. In some embodiments, a reverse primer of the second primer set will have at least 1 , at least 2 or at least 3 complementary bases to the first molecule within the second 10 bases of the primer. Each possibility represents a separate embodiment of the invention.
  • qPCR Quantitative PCR
  • duplex PCR refers to a single reaction, done in a single vessel but containing two amplification reactions.
  • a multiplex PCR is thus a single reaction, done in a single vessel but containing more than two amplifications.
  • the methods of the invention are performed wherein the qPCR is a multiplex PCR and further comprises at least one additional set of primers with at least 70% complementarity to a target nucleic acid molecule and 10-99% complementarity to a control nucleic acid molecule, and wherein no nucleic acid molecule has greater than 10% complementarity to more than two sets of primers.
  • the methods of the invention are performed wherein the qPCR is a multiplex PCR and further comprises at least one additional set of primers with at least 70% complementarity to a target nucleic acid molecule, at least 10% complementarity to a control nucleic acid molecule, and greater complementarity to the target nucleic acid molecule than to the control nucleic acid molecule, and wherein no nucleic acid molecule has greater than 10% complementarity to more than two sets of primers.
  • the reagents, primers, polymerase, probes, ions, etc. must be shared amongst the amplifications.
  • the duplex PCR of the invention is any PCR in which two amplifications can be performed at once in a single vessel with shared reagents.
  • the duplex PCR of the invention is Taqman qPCR. It will be understood by a skilled artisan that the percentages of complementarity described above for the first and set sets of primers will also apply for this additional set of primers. All above recited ranges and value will also apply to this additional set of primers.
  • Kits for performing such include but are not limited to the TaqMan Universal PCR Master Mix, the QIAGEN multiplex PCR kit, and the Thermo Fisher Platinum Multiplex PCR Master Mix.
  • Kits for performing reverse transcription of RNA to cDNA include, for non-limiting example, the Cells- to-cDNA kit, the ProtoScript First Strand cDNA synthesis kit, and the Superscript VIFO cDNA synthesis kit.
  • the qPCR reactions of the invention may be performed on any system known in the art for performing such.
  • thermal cyclers for use in performing the reactions of the invention include, but are not limited to, the QuantStudio Real-Time PCR systems from Thermo Fischer, the ABI 7000/7300/7500 Real-Time PCR systems and the CFX line of Real-Time PCR detection systems from Bio-Rad.
  • the software that accompanies these systems may be used for the calculation described herein that pertain to determining threshold values, cycle values, and relative expression. Further, statistical software and Microsoft Excel may be used for calculations of Ct differences, error, and regression curves such as are described herein.
  • At least one reagent of the qPCR reaction is present in a low amount.
  • a low amount is an amount below the amount recommended by a manufacturer of the reagent.
  • the at least one reagent is present at less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amount or concentration recommended by the manufacturer.
  • the low amount of at least one of the primers is between 50 and 500, 50 and 450, 50 and 400, 50 and 350, 50 and 300, 50 and 250, 50 and 200, 50 and 150, 100 and 500, 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, or 100 and 150 nM per reaction.
  • at least one of the primers is present at between 100 and 300 nM per reaction. In some embodiments, at least one of the primers is present at about 150 nM per reaction. In some embodiments, all primers are present at between 100 and 300 nM per reaction. In some embodiments, all primers are present at about 150 nM per reaction.
  • the low amount of a qPCR Mastermix is 75% of an amount recommended by a manufacturer. In some embodiments, the low amount of a 2X qPCR Mastermix is 37.5% of the final volume of the reaction. Thus, a reaction performed in a final volume of 50 ul, which would, as recommended by the manufacturer, have 25 ul of 2X qPCR Mastermix, will instead have 18.75 ul.
  • the qPCR reaction comprises a first probe for the first set of primers and a second probe for the second set of primers.
  • the first probe has 90- 100% complementarity to the amplification product of the first set of primers and 15-99% complementarity to the amplification product of the second set of primers.
  • the second probe has 90-100% complementarity to the amplification product of the second set of primers and 15-99% complementarity to the amplification product of the first set of primers.
  • the first probe comprises greater complementarity to the first amplification product than to the second amplification product.
  • the second probe comprises greater complementarity to the second amplification produce than to the first amplification product.
  • the qPCR reaction comprises a probe
  • at least one probe is present at between 50 and 500, 50 and 450, 50 and 400, 50 and 350, 50 and 300, 50 and 250, 50 and 200, 50 and 150, 100 and 500, 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, or 100 and 150 nM per reaction.
  • the probes is present at between 100 and 300 nM per reaction. In some embodiments, at least one of the probes is present at about 150 nM per reaction.
  • all probes are present at between 100 and 300 nM per reaction. In some embodiments, all probes are present at about 150 nM per reaction. In some embodiments, both the first probe and the second probe are preset at about 150 nM or between 100 and 300 nM per reaction.
  • the reaction comprises between 10 L 1 copies and 10 L 8 copies of the target nucleic acid molecule. In some embodiments, the reaction comprises between 10 L 2 copies and 10 L 6 copies of the target nucleic acid molecule. In some embodiments, the first sample comprises at least 10 L 3 copies of the target nucleic acid molecule. In some embodiments, the first sample comprises about 10 L 3 copies of the target nucleic acid molecule. In some embodiments, the first sample comprises between 50-500, 50-450, 50-400, 50-350, 50-300, 100-500, 100-450, 100-400, 100-350, 100-300 ug of DNA. Each possibility represents a separate embodiment of the invention. In some embodiments, the reaction comprises about 160 ug of DNA.
  • At least one cycle of the qPCR has a reduced run time as compared to the time recommended by a manufacturer.
  • the manufacturer is the manufacturer of the PCR Mastermix.
  • the manufacturer is the manufacturer of the PCR machine or thermal cycler.
  • at least one cycle of a qPCR has a reduced annealing and/or extending time as compared to the annealing and/or extending time recommended by a manufacturer.
  • at least one cycle of a qPCR has a reduced denaturing time as compared to the denaturing time recommended by a manufacturer.
  • the time is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
  • the denaturing time is between 2 and 8 seconds. In some embodiments, the denaturing time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some embodiments, the denaturing time is about 4 seconds.
  • the annealing and/or extending time is between 10 and 30 seconds. In some embodiments, the annealing and/or extending time is at most 10, 15, 20, 25, or 30 seconds. Each possibility represents a separate embodiment of the invention. In some embodiments, the annealing and/or extending time is about 10, 15, 20, 25, or 30 seconds. Each possibility represents a separate embodiment of the invention.
  • PCR reaction parameters may vary depending on the reagents, purity of the starting sample, and thermocycler used.
  • a standard duplex PCR as recommended by the manufacturer comprises at least 35 cycles of a denaturing step at 95 degrees for 15 seconds and an annealing/extending step at 60 degrees for 60 seconds.
  • all the cycles of the PCR run have a reduced denaturing time, annealing/extending time or both.
  • the small difference in an amount is less than a 0.1%, 0.2%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% change in the amount.
  • the small difference in an amount is between a 10% and 0.2%, 5% and a 0.2%, 10% and 0.5%, 5% and a 0.5%, 10% and 1%, or 5% and a 1%, change in the amount.
  • the small difference in amount is less than can be measured in one cycle, half a cycle, or a quarter of a cycle of a duplex PCR.
  • Each possibility represents a separate embodiment of the invention.
  • the methods of the invention further comprise performing duplex qPCR with the first and second set of primers on nucleic acids from the second sample and measuring API and AP2 in the second sample.
  • the second sample is a control sample.
  • the second sample is a control sample and the ratio of API to AP2 in the control sample is known.
  • the methods of the invention further comprise subtracting the ACt for the first nucleic acid molecule from the ACt for the second nucleic acid molecule to receive a total change in the amount of the first nucleic acid molecule. As demonstrated herein, the difference in the ACt is proportionate to the total change in the amount of the first nucleic acid molecule.
  • a standard curve is created from control samples to determine how the change in ACt correlates to the change in amount of the first nucleic acid molecule.
  • the first nucleic acid molecule is the target molecule.
  • the second nucleic acid molecule is the control or standard molecule.
  • the second nucleic acid molecule is present in the same abundance or concentration in the first sample and the second sample.
  • Control and standard molecules are well known in the art and may be a house keeping gene such as gapdh, actin, ribosomal RNA or the like. The control molecule may be selected based on what the target molecule is, and the source of the nucleic acid molecules being tested. If for instance genomic DNA is being tested, a genomic locus that is invariant may be selected, whereas if cDNA reverse transcribed from RNA is being tested a house keeping gene maybe selected.
  • control sequence from the host or subject may be used as the control.
  • the control is a control chromosome.
  • the control chromosome is a chromosome that does not bare a genetic mutation.
  • the control chromosome is a chromosome with invariability in its number.
  • a chromosome with invariability in number is any chromosome other than 8, 9, 13, 18 and 21.
  • a chromosome with invariability in number is any chromosome other than 8, 9, 13, 18, 21 and 22.
  • the control is a sex chromosome.
  • the control is the X chromosome.
  • control is a locus on the chromosome.
  • the nucleic acids from the first sample are any one of cDNA reverse transcribed from RNA, and genomic DNA (gDNA).
  • the nucleic acids are gDNA.
  • the nucleic acids are cDNA.
  • the methods of the invention further comprise reverse transcribing RNA into cDNA before performance of the method.
  • the genomic DNA is fetal genomic DNA.
  • the nucleic acids are cell free DNA (cfDNA).
  • the cfDNA is cell free fetal DNA (cffDNA).
  • the cfDNA is circulating tumor DNA.
  • the cfDNA is a mix of maternal cfDNA and cffDNA.
  • “cfDNA” refers to DNA that is not inside a cell, but rather is free available in a biological fluid. Non-limiting examples of such include DNA from tumors which can be found floating freeing in the blood and fetal DNA which can be found in the mother’s blood (cffDNA).
  • the first nucleic acid molecule comprises a disease associated mutation.
  • disease associated mutations refers to a DNA or RNA sequence which can be used to diagnose a disease or disorder.
  • Disease associated mutations maybe known cancer causing mutations, mutations known to cause hereditary disease, or known deletions, insertions or duplication known to cause disease. Examples of such, include but are not limited to Trisomy of chromosome 13, point mutation of the dystrophin gene or CF mutations in cystic fibrosis.
  • the first nucleic acid molecule is an oncogene.
  • Oncogenes are well known in the art.
  • the first sample is a biopsy.
  • the first nucleic acid molecule is a cancer driver.
  • the disease associated mutation is a driver mutation. Examples of oncogenes and cancer driving mutations include, but are not limited to, KRAS, p53 mutations, mutations of BRCA1 and BRCA2.
  • the first nucleic acid molecule is a molecule that comprises a marker for a genetic disorder. In some embodiments, the first nucleic acid molecule is a marker for a genetic disorder. In some embodiments, the genetic disorder is caused by a mutation, an insertion, a deletion or a duplication. In some embodiments, the genetic disorder is a trisomy of a chromosome. In some embodiments, the trisomy is trisomy of any one of chromosome 8, 9, 13, 18 and 21. In some embodiments, the trisomy is trisomy of chromosome 21. In some embodiments, the genetic disorder is Down syndrome.
  • the first sample is a sample contaminated with a foreign or unknown nucleic acid.
  • the first sample is a sample at risk of being contaminated.
  • the first sample is a sample suspected of being contaminated.
  • the first nucleic acid molecule is the foreign or unknown nucleic acid.
  • the foreign or unknown nucleic acid comprises DNA or RNA from a pathogen.
  • the pathogen is selected from a virus, a bacterium and a fungus.
  • the foreign or unknown nucleic acid is DNA or RNA from an unknown human.
  • the first sample is a sample to be tested and the second sample is a control sample.
  • the sample to be tested is from a fetus.
  • the sample to be tested is form a pregnant subject.
  • the sample to be tested comprises maternal and fetal nucleic acids.
  • the sample is from a fetus or comprises fetal nucleic acids and the first nucleic acid molecule is a marker for a genetic disorder.
  • the fetus is at risk of having a genetic disorder.
  • the fetus is suspected of having a genetic disorder.
  • the sample to be tested is from a subject at risk of developing cancer or who has already developed cancer. In some embodiments, the sample is from a subject at risk of developing cancer and the first nucleic acid molecule is a cancer marker, oncogene or comprises a driver mutation. In some embodiments, the sample to be tested is from a biopsy. In some embodiments, the sample to be tested is from a liquid biopsy. In some embodiments, the sample to be tested is from drawn blood. In some embodiments, the sample to be tested is possibly contaminated. In some embodiments, the contamination is from a human. In some embodiments, the contamination is from a pathogen. In some embodiments, the pathogen is selected from a virus, a bacterium and a fungus.
  • the term“contaminated” refers to the presence of nucleic acids from a secondary source within the primer source of the sample.
  • contamination include but are not limited to viral DNA or RNA in a human sample, bacterial DNA in a human sample, DNA from one human in a sample from another human.
  • the methods of the invention can be used for detection of small amounts of nucleic acids they can be employed to detect a small level of contamination that might not be detectable with conventional qPCR.
  • the genetic disorder is a hereditary disorder. In some embodiments, the genetic disorder is a chromosomal duplication. In some embodiments, the genetic disorder is at a locus and the target nucleic acid is that locus. Examples of genetic disorders include, but are not limited to, Down syndrome, cystic fibrosis, sickly cell anemia, Tay-Sachs disease, Marfan syndrome, Edward’s syndrome, Patau syndrome, Cri du chat syndrome, Wolf-Hirschhorn syndrome, Jacobsen syndrome, Klinefelter’s syndrome, and Turner syndrome. In some embodiments, the genetic disorder is a chromosomal abnormality. In some embodiments, the genetic disorder comprises a chromosomal abnormality.
  • the genetic disorder is characterized by a chromosomal abnormality.
  • the genetic disorder is caused by a chromosomal abnormality.
  • the abnormality is a chromosomal duplication.
  • the abnormality is a deletion.
  • the abnormality is in a region of a chromosome.
  • the abnormality is of a whole chromosome.
  • the chromosomal abnormality is selected from Down syndrome, Edward’s syndrome, Patau syndrome, Cri du chat syndrome, Wolf-Hirschhorn syndrome, Jacobsen syndrome, Klinefelter’s syndrome, and Turner syndrome.
  • the chromosomal abnormality is Down syndrome. Kits
  • kits comprising two sets of primers, wherein a first set of primers comprises at least 90% complementarity to a target sequence of interest, and 10-99% complementarity to a control sequence, and wherein a second set of primers comprises at least 90% complementarity to the control sequence and 10-99% complementarity to the target sequence.
  • kits comprising two sets of primers, wherein a first set of primers comprises at least 90% complementarity to a target sequence of interest, at least 10% complementarity to a control sequence, and greater complementarity to the target sequence than to the control sequence and wherein a second set of primers comprises at least 90% complementarity to the control sequence, at least 10% complementarity to the target sequence and greater complementarity to the control sequence than to the target sequence.
  • the first set of primers comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity to a target sequence of interest. Each possibility represents a separate embodiment of the invention. In some embodiments, the first set of primers comprises 100% complementarity to a target sequence of interest. In some embodiments, the second set of primers comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity to a control sequence. Each possibility represents a separate embodiment of the invention. In some embodiments, the second set of primers comprises 100% complementarity to a control sequence. In some embodiments, the first set of primers is the first set of primers described hereinabove. In some embodiments, the second set of primers is the second set of primers described herein above.
  • the kit further comprises at least one reagent for qPCR.
  • the at least one reagent is for duplex qPCR and/or multiplex qPCR.
  • the at least one reagent is qPCR Mastermix.
  • the kit further comprises a first probe for the first set of primers and a second probe for the second set of primers.
  • the first probe comprises 90-100% complementarity to the amplification product of the first set of primers and 10-99% complementarity to the amplification product of the second set of primers.
  • the first probe comprises at least 90% complementarity to the amplification product of the first set of primers, at least 10% complementarity to the amplification product of the second set of primers, and greater complementarity to the amplification product of the first set of primers than to the amplification product of the second set of primers.
  • the probe for the second set of primers has 90-100% complementarity to the amplification product of the second set of primers and 10- 99% complementarity to the amplification product of the first set of primers.
  • the second probe comprises at least 90% complementarity to the amplification product of the second set of primers, at least 10% complementarity to the amplification product of the first set of primers, and greater complementarity to the amplification product of the second set of primers than to the amplification product of the first set of primers.
  • kits of the invention further comprise instructions for use.
  • the instructions comprise at least one of the thermocycling conditions, the method of calculating Ct values, calculations for determining the difference in Ct values, methods of converting differences in Ct values to differences in expression of the target molecule, a standard table or curve with values for converting differences in Ct values to changes in expression or amounts of a target molecule.
  • kits of the of the invention are for use in performing a method of the invention. In some embodiments, the kits of the of the invention are for use in detecting or measuring small differences in an amount of the target sequence of interest in a test sample as compared to a control sample. In some embodiments, the kit further comprises a table of values of PCR results or Ct values in at least one control sample. In some embodiments, the kit further comprises a table of differences in Ct value and the amount of change in the target sequence of interest that correspond to those differences.
  • kits of the invention are for use in cancer diagnosis. In some embodiments, the kits of the invention are for use in determining the driver or oncogenic cause of a cancer. In some embodiments, the kits of the invention are for use in prenatal diagnosis of a fetus. In some embodiments, the kits of the invention are for use in non-invasive prenatal diagnosis (NIPD). In some embodiments, the kits of the invention are for use in genetic testing. In some embodiments, the kits of the invention are for use it detecting or classifying a pathogen. As used herein,“classifying a pathogen” refers to any form of differentiating one pathogen from another.
  • kits of the invention are for use in detecting small changes in gene expression.
  • the kits of the invention are for use in forensic science. It may be important to identify from which individual a sample originated, or to detect a contaminating amount of DNA from one person in a sample from another person, and the kit and methods of the invention can be used for such.
  • the kits of the invention are for use in precise organism counting. It can be difficult to precisely count viral genomes or bacteria, and the kits and methods of the invention may be used for such.
  • the kits of the invention are for use in detection of species variation.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • a modified Taqman duplex qPCR was then performed with these primers and the control gDNA (XY) and mixed (XY+ 5%X) gDNA.
  • reagents were kept at a low amount, significantly below what is recommended by the manufacturer. Reactions were performed in a final volume of 25 ul with the following amounts of reagents:
  • Taqman probe 150 nM
  • DNA 10 L 3 copies of control target, 160 ug total 2X master mix: 9.375 ul (75% of recommended amount)
  • cfDNA Cell free DNA
  • cffDNA refers to free floating DNA fragments that can be found in all blood. cfDNA is believed to originate, at least in part, from cells that have undergone apoptosis and released their DNA into circulation. A specific subclass of cfDNA is cell free fetal DNA (cffDNA). cffDNA is DNA from fetal cells that can be detected in the blood of the mother. Maternal blood is estimated to contain between 3-10% cffDNA and 90-97% maternal cfDNA. For the purposes of the herein described experiments, cffDNA percentage is approximated as 6%. The following experiments exemplify the use of competitive duplex qPCR for measuring very small differences in DNA in a naturally occurring setting.
  • Down syndrome is characterized by trisomy of chromosome 21.
  • a noninvasive test for trisomy by measuring cffDNA is hampered by the fact that the vast majority of cfDNA used for an assay is from the mother (who has only 2 copies of chromosome 21).
  • 94% of the chromosome 21 will be maternal in origin and 6% will be fetal in origin.
  • a mother carrying an fetus with Down syndrome there will 1.5 times the amount of fetal chromosome 21. Therefore, there will be 3% more total chromosome 21 in the cfDNA. The challenge is thus to consistently and accurately detect changes as small as 3%.
  • the above described primers for measuring the X chromosome were also used for this experiment as the primers had -20% complementarity to chromosome 21.
  • the primers for amplifying chromosome 21, which also had low complementarity to the X chromosome, are provided in Table 2.
  • Primer were designed to amplify chromosome 21 with 100% complementarity to their target (GGGTCGGCTCTGCAATAAACTGTAACTGTGAGTTTACTGGTTCCTGAGCTCTGCAAG TCCTTCCGGCAAATCACTGATCCTGAGAAAGGTCTTGG, SEQ ID NO: 12).
  • Taqman probe 150 nM
  • DNA ⁇ 10 L 3 copies of control target, 4 ul total
  • ddH20 fill up to 25 ul total
  • the following PCR cycle was run on a STEP-1 PCR system, which included both shorter denaturing and annealing/extension times as compared to the manufacturer’s recommendation:

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Abstract

Procédés et kits pour détecter et mesurer de petites différences dans des quantités d'une séquence d'acide nucléique cible dans un échantillon test par comparaison avec un échantillon témoin à l'aide de PCR quantitative (qPCR) duplex compétitive.
PCT/IL2019/051286 2018-11-25 2019-11-25 Procédés de mesure de petites différences d'abondance d'acides nucléiques Ceased WO2020105058A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010088288A2 (fr) * 2009-01-28 2010-08-05 Fluidigm Corporation Détermination de différences du nombre de copies par amplification
CN104988239A (zh) * 2015-08-06 2015-10-21 上海市第一妇婴保健院 一种快速分析单细胞性染色体倍性的方法和试剂盒
WO2017190106A1 (fr) * 2016-04-29 2017-11-02 Medical College Of Wisconsin, Inc. Nombre de cibles par amplification de mésappariement (moma) optimisée multiplexée (moa)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010088288A2 (fr) * 2009-01-28 2010-08-05 Fluidigm Corporation Détermination de différences du nombre de copies par amplification
CN104988239A (zh) * 2015-08-06 2015-10-21 上海市第一妇婴保健院 一种快速分析单细胞性染色体倍性的方法和试剂盒
WO2017190106A1 (fr) * 2016-04-29 2017-11-02 Medical College Of Wisconsin, Inc. Nombre de cibles par amplification de mésappariement (moma) optimisée multiplexée (moa)

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