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WO1999029905A2 - Methode de detection de l'apoptose a l'aide d'oligonucleotides marques par fret - Google Patents

Methode de detection de l'apoptose a l'aide d'oligonucleotides marques par fret Download PDF

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Publication number
WO1999029905A2
WO1999029905A2 PCT/US1998/026432 US9826432W WO9929905A2 WO 1999029905 A2 WO1999029905 A2 WO 1999029905A2 US 9826432 W US9826432 W US 9826432W WO 9929905 A2 WO9929905 A2 WO 9929905A2
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Prior art keywords
die
strand
oligonucleotide
labeled
nucleotide sequence
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PCT/US1998/026432
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English (en)
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WO1999029905A3 (fr
WO1999029905B1 (fr
Inventor
William M. James
Irina A. Nazarenko
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Intergen Company
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Priority to JP2000524476A priority Critical patent/JP2001526052A/ja
Priority to CA002314225A priority patent/CA2314225A1/fr
Priority to EP98963108A priority patent/EP1036201A2/fr
Publication of WO1999029905A2 publication Critical patent/WO1999029905A2/fr
Publication of WO1999029905A3 publication Critical patent/WO1999029905A3/fr
Publication of WO1999029905B1 publication Critical patent/WO1999029905B1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6862—Ligase chain reaction [LCR]

Definitions

  • the present invention is broadly concerned with methods for detecting target nucleic acid fragments such as those generated during the process of apoptosis. More particularly, these methods are ligase-mediated polymerase chain reaction (LM-PCR) methods in which an oligonucleotide is incorporated into the amplification product of the target nucleic acid fragment, wherein the oligonucleotide is labeled with a moiety that emits detectable energy only if the oligonucleotide is incorporated into the amplification product; detection of the energy emitted by the moiety thereby indicates that the target nucleic acid fragment has been amplified.
  • LM-PCR ligase-mediated polymerase chain reaction
  • apoptosis also called cellular suicide or programmed cell death.
  • apoptosis also called cellular suicide or programmed cell death.
  • apoptosis and mitosis are engaged in a homeostatic balance in which an appropriate tissue mass is maintained.
  • Disorders of apoptosis are believed to be among the causes of cancer and many other kinds of pathologies. Therefore, apoptosis is a prime target for therapeutic intervention.
  • nucleic acids are hydrolyzed by cellular nucleases.
  • DNA is wound around packets of DNA-binding proteins to form structural units called nucleosomes.
  • DNA within nucleosomes is partially protected from nucleolytic attack. Since nucleosomes are spaced along a DNA molecule at regular intervals of 180 to 200 bp, nuclease cuts occur between nucleosomes at similar intervals. As a result, a series of DNA fragments (called an "oligo-nucleosomal DNA ladder”) can be visualized when DNA hydrolyzed in this manner is separated by electrophoresis in an analytical agarose gel.
  • oligo-nucleosomal DNA ladder a series of DNA fragments
  • This ladder in which the fragment lengths are integral multiples of from 180 to 200 bp, is widely regarded as a biochemical hallmark of apoptosis.
  • apoptosis normally affects only a very small percentage of cells in a tissue. But sustained apoptosis can rapidly cause a tissue mass to decline. Thus, very sensitive detection methods are required to quantitate these low but significant levels of apoptosis.
  • the large number of DNA ends in the oligo-nucleosomal DNA ladder provides an opportune marker for scarce cells undergoing apoptosis. Staley et al., 1997, Cell Death & Differen ⁇ ation 4:66-75, describe a method for detecting these DNA ends.
  • Staley et al. adapted the method of LM-PCR for the amplification of apoptotic nucleosomal DNA fragments (see Figure 8).
  • the amplified DNA fragments are quantitated using a gel electrophoretic assay in which sample band densities are compared.
  • the gel electrophoresis assay of Staley et al. is labor- and time- intensive. Accordingly, there is a need in the art for improved methods of detecting DNA fragments resulting from the process of apoptosis.
  • the present invention is directed to LM-PCR methods for detecting DNA fragments resulting from the process of apoptosis.
  • a label is incorporated directly into the amplification product, making the amplification product immediately detectable without opening the reaction vessel.
  • the detection system of the present invention makes use of the molecular energy transfer (MET) phenomenon.
  • MET is a process by which energy is passed between a donor molecule and an acceptor molecule.
  • Fluorescence resonance energy transfer (FRET) is a form of MET. FRET arises from the properties of certain chemical compounds termed fluorophores. When a fluorophore is excited by exposure to a particular wavelength of light, it emits light (fluoresces) at a different wavelength.
  • FRET energy is passed between a donor molecule, which is a fluorophore, and an acceptor molecule. The donor molecule absorbs a photon and transfers this energy to the acceptor molecule. F ⁇ rster, 1949, Z. Naturforsch A4: 321-327; Clegg, 1992, 211 METHODS IN
  • MET pairs or FRET pairs Pairs of molecules that can engage in MET or FRET are termed MET pairs or FRET pairs, respectively.
  • excitation of one fluorophore will cause it to emit light at a wavelength that is absorbed by and that stimulates the second fluorophore, causing it in turn to fluoresce.
  • the fluorescence of the donor molecule is quenched, while the fluorescence of the acceptor molecule is enhanced.
  • the excited-state energy of the donor is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quencher.
  • the present invention utilizes oligonucleotides which are incorporated by LM-PCR amplification into DNA fragments, for example, resulting from the process of apoptosis.
  • Each oligonucleotide contains an acceptor moiety and/or a donor moiety, and therefore is referred to as a "FRET-labeled oligonucleotide.”
  • the acceptor and donor moieties are situated on different nucleotide sequences, wherein the nucleotide sequences are either in a single oligonucleotide or in separate oligonucleotides.
  • the acceptor and donor moieties When these nucleotide sequences are annealed to each other, the acceptor and donor moieties are in close proximity, and the acceptor moiety absorbs the energy emission from the donor moiety, thereby quenching the signal from the donor moiety. When the nucleotide sequences are not annealed to each other, however, the acceptor and donor moieties are separated, the acceptor moiety can no longer absorb the energy emission from the donor moiety, and the signal from the donor is not quenched. Thus, detection of a signal indicates that the two nucleotide sequences are not annealed to each other.
  • the nucleotide sequence containing the donor moiety is no longer annealed to the nucleotide sequence containing the acceptor moiety. Consequently, the acceptor and donor moieties are separated from each other, and the acceptor moiety no longer quenches the signal from the donor moiety, resulting in the generation of an observable signal. This signal indicates that the double-stranded DNA fragment of interest has been amplified.
  • FRET-labeled oligonucleotides are described in PCT application WO 98/02449, published January 22, 1998.
  • LM-PCR conducted using FRET-labeled oligonucleotides is referred to as "fluorescent LM-PCR" (FLM-PCR).
  • FLM-PCR fluorescent LM-PCR
  • the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
  • a linker-primer duplex which contains a first oligonucleotide (a linker-primer oligonucleotide) and second oligonucleotide (a ligation-aid oligonucleotide), wherein the first oligonucleotide is annealed to the second oligonucleotide.
  • a third oligonucleotide (a detectable MET-labeled oligonucleotide, e.g., a FRET-labeled oligonucleotide) which contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair.
  • a detectable MET-labeled oligonucleotide e.g., a FRET-labeled oligonucleotide
  • the molecular energy transfer pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first nucleotide sequence is capable of annealing to the complement of the first oligonucleotide.
  • the second nucleotide sequence contains the donor moiety and the fourth nucleotide sequence contains the acceptor moiety, or the second nucleotide sequence contains the acceptor moiety and the fourth nucleotide sequence contains the donor moiety.
  • the second nucleotide sequence is annealed to the fourth nucleotide sequence to form a hai ⁇ in.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the hai ⁇ in is formed.
  • step (b) the 3' end of the first oligonucleotide is ligated to the 5' end of the first strand of the fragment to form a ligated first strand, and the 3' end of the first oligonucleotide is ligated to the 5' end of the second strand of the fragment to form a ligated second strand.
  • step (c) the 3' end of the ligated first strand is extended using the ligated second strand as a template to form an extended first strand. Additionally, the 3' end of the ligated second strand is extended using the ligated first strand as a template to form an extended second strand. Consequently, the extended first strand is annealed to the extended second strand after these extension reactions.
  • step (d) the extended first strand is separated from the extended second strand.
  • step (e) the third oligonucleotide is annealed to each of the extended first and second strands.
  • step (f) the 3' end of the third oligonucleotide is extended using the extended first strand as a template to form a labeled second strand, and the 3' end of the extended first strand is extended using the third oligonucleotide as a template to form a doubly extended first strand.
  • the 3' end of the third oligonucleotide is extended using the extended second strand as a template to form a labeled first strand
  • the 3' end of the extended second strand is extended using the third oligonucleotide as a template to form a doubly extended second strand. Consequently, the labeled second strand is annealed to the doubly extended first strand, and the labeled first strand is annealed to the doubly extended second strand after these extension reactions.
  • step (g) the labeled second strand is separated from the doubly extended first strand, and the labeled first strand is separated from the doubly extended second strand.
  • step (h) the third oligonucleotide is annealed to each of the labeled first and second strands, and to each of the doubly extended first and second strands.
  • step (i) the 3' end of the third oligonucleotide is extended using the labeled first strand and the doubly extended first strand as templates to form extended labeled second strands. Additionally, the 3' end of the third oligonucleotide is extended using the labeled second strand and the doubly extended second strand as templates to form extended labeled first strands.
  • step (j) which is optional, the extended labeled first and second strands are amplified.
  • step (k) which follows either step (f), (i), or (j), the emitted energy (e.g., light) is detected in order to detect the fragment.
  • the emitted energy e.g., light
  • step (j) a variety of amplification methods can be used in step (j) to amplify the extended labeled first and second strands (e.g., PCR amplification, strand displacement amplification, and cascade rolling circle amplification).
  • the preferable method of amplification is PCR amplification comprising the following four steps:
  • step (i) the extended labeled first strand is separated from the extended labeled second strand.
  • step (ii) the third oligonucleotide is annealed to each of the extended labeled first and second strands.
  • step (iii) the 3' end of the third oligonucleotide is extended using the extended labeled first strand as a template to form another extended labeled second strand, wherein the extended labeled first strand is annealed to the other extended labeled second strand. Additionally, the 3' end of the third oligonucleotide is extended using the extended labeled second strand as a template to form another extended labeled first strand, wherein the extended labeled second strand is annealed to the other extended labeled first strand.
  • steps (i), (ii), and (iii) are repeated for a finite number of times, wherein, in (i), the extended labeled first and second strands respectively are the extended labeled first strand and the other extended labeled second strand of (iii), or respectively are the other extended labeled first strand and the extended labeled second strand of (iii).
  • the donor moiety is a fluorophore and the acceptor moiety is a quencher of light emitted by the fluorophore.
  • the donor moiety may be fluorescein, 5- carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate, and Reactive Red 4.
  • the acceptor moiety may be DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, fluorescein, Malachite green, or Texas Red.
  • the donor moiety is fluorescein
  • the acceptor moiety is DABCYL
  • the donor and acceptor moieties are respectively attached to complementary first and second nucleotides in the hai ⁇ in so that the acceptor moiety is in close proximity to the donor moiety.
  • the 3' end of the second oligonucleotide preferably contains a blocking group that prevents this end from being extended in a reaction in which the first oligonucleotide acts as a template.
  • a phosphate group preferably is not attached to the 3' end of the first oligonucleotide, which allows this end to be ligated to each strand of the fragment if each strand contains a phosphate group at the 5' end.
  • a phosphate group preferably is not attached to the 5' end of the second oligonucleotide, which prevents this end from being ligated to the 3' end of the first oligonucleotide, thereby preventing self- ligation of the linker-primer duplex.
  • the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands, which allows the latter end to be extended by a nucleic acid polymerase.
  • the third oligonucleotide consists of the sequence 5'-AGCTGGAACTCTATCCAGCTACTGAACCTGACCGTACA-3' (SEQ ID NO:l), wherein fluorescein is attached to the residue at position 1 and DABCYL is attached to the residue at position 20, and the second oligonucleotide consists of a sequence selected from the group consisting of 5'-TGTACGGTCAGG-3' (SEQ ID NO:2), 5'- ATGTACGGTCAGG-3' (SEQ ID NO:3), 5'-TTGTACGGTCAGG-3' (SEQ ID NO:4), 5'-CTGTACGGTCAGG-3' (SEQ ID NO:5), and 5XGTGTACGGTCAGG-3' (SEQ ID NO:6).
  • an end of the fragment is a blunt end
  • the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a blunt end in the duplex.
  • an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases)
  • the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.
  • the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
  • a linker-primer duplex containing a first oligonucleotide (a linker- primer/MET-labeled oligonucleotide) and a second oligonucleotide (a ligation-aid oligonucleotide) is provided, wherein the first oligonucleotide contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair.
  • the molecular energy transfer pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first nucleotide sequence is annealed to the second oligonucleotide.
  • the second nucleotide sequence contains the donor moiety and the fourth nucleotide sequence contains the acceptor moiety, or the second nucleotide sequence contains the acceptor moiety and the fourth nucleotide sequence contains the donor moiety.
  • the second nucleotide sequence is annealed to the fourth nucleotide sequence to form a hai ⁇ in.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the hai ⁇ in is formed.
  • step (b) the 3' end of the first oligonucleotide is ligated to the 5' end of the first strand of the fragment to form a labeled first strand, and the 3' end of the first oligonucleotide is ligated to the 5' end of the second strand of the fragment to form a labeled second strand.
  • step (c) the 3' end of the labeled first strand is extended using the labeled second strand as a template to form an extended labeled first strand. Additionally, the 3' end of the labeled second strand is extended using the labeled first strand as a template to form an extended labeled second strand. Consequently, the extended labeled first strand is annealed to the extended labeled second strand after these extension reactions.
  • step (d) which is optional, the extended labeled first and second strands are amplified.
  • step (e) which follows step (b), (c), or (d), the emitted energy (e.g., light) is detected in order to detect the fragment.
  • amplification methods can be used in step (d) to amplify the extended labeled first and second strands (e.g., PCR amplification, strand displacement amplification, and cascade rolling circle amplification).
  • the preferable method of amplification is PCR amplification comprising the following four steps:
  • step (i) the extended labeled first strand is separated from the extended labeled second strand.
  • step (ii) the first oligonucleotide is annealed to each of the extended labeled first and second strands.
  • step (iii) the 3' end of the first oligonucleotide is extended using the extended labeled first strand as a template to form another extended labeled second strand, wherein the extended labeled first strand is annealed to the other extended labeled second strand. Additionally, the 3' end of the first oligonucleotide is extended using the extended labeled second strand as a template to form another extended labeled first strand, wherein the extended labeled second strand is annealed to the other extended labeled first strand.
  • steps (i), (ii), and (iii) are repeated for a finite number of times, wherein, in (i), the extended labeled first and second strands respectively are the extended labeled first strand and the other extended labeled second strand of (iii), or respectively are the other extended labeled first strand and the extended labeled second strand of (iii).
  • a phosphate group preferably is not attached to d e 3' end of the first oligonucleotide, which allows this end to be ligated to each strand of the fragment if each strand contains a phosphate group at the 5' end.
  • a phosphate group preferably is not attached to the 5' end of the second oligonucleotide, which prevents this end from being ligated to the 3' end of the first oligonucleotide, thereby preventing self-ligation of the linker-primer duplex.
  • the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands, which allows the latter end to be extended by a nucleic acid polymerase. If an end of the fragment is a blunt end, then the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a blunt end in the duplex.
  • an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases)
  • a terminal overhang e.g., a terminal overhang of 1 to 10 bases
  • the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.
  • the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
  • a linker-primer oligonucleotide (which also acts as a ligation-aid/MET- labeled oligonucleotide) is provided which contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, and (iv) a molecular energy transfer pair.
  • the molecular energy transfer pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first nucleotide sequence is annealed to the third nucleotide sequence to form a hai ⁇ in.
  • the first nucleotide sequence contains the 3' end of the oligonucleotide.
  • the third nucleotide sequence contains the 5' end of the oligonucleotide.
  • the first nucleotide sequence contains the donor moiety and the third nucleotide sequence contains the acceptor moiety, or the first nucleotide sequence contains the acceptor moiety and the third nucleotide sequence contains the donor moiety.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the hai ⁇ in is formed.
  • step (b) the 3' end of the oligonucleotide is ligated to the 5' end of the first strand of the fragment to form a labeled first strand, and the 3' end of the oligonucleotide is ligated to the 5' end of the second strand of the fragment to form a labeled second strand.
  • step (c) the 3' end of the labeled first strand is extended using the labeled second strand as a template to form an extended labeled first strand. Additionally, the 3' end of the labeled second strand is extended using the labeled first strand as a template to form an extended labeled second strand. Consequently, the extended labeled first strand is annealed to the extended labeled second strand after these extension reactions.
  • step (d) which is optional, the extended labeled first and second strands are amplified.
  • step (e) which follows step (b), (c), or (d)
  • the emitted energy e.g., light
  • step (d) the emitted energy (e.g., light) is detected in order to detect the fragment.
  • a variety of amplification methods can be used in step (d) to amplify the extended labeled first and second strands (e.g., PCR amplification, strand displacement amplification, and cascade rolling circle amplification).
  • the preferable method of amplification is PCR amplification comprising the following four steps: In step (i), the extended labeled first strand is separated from the extended labeled second strand.
  • step (ii) the oligonucleotide is annealed to each of the extended labeled first and second strands.
  • step (iii) the 3' end of the oligonucleotide is extended using the extended labeled first strand as a template to form another extended labeled second strand, wherein the extended labeled first strand is annealed to the other extended labeled second strand. Additionally, the 3' end of the oligonucleotide is extended using the extended labeled second strand as a template to form another extended labeled first strand, wherein the extended labeled second strand is annealed to the other extended labeled first strand.
  • steps (i), (ii), and (iii) are repeated for a finite number of times, wherein, in (i), the extended labeled first and second strands respectively are the extended labeled first strand and the other extended labeled second strand of (iii), or respectively are the other extended labeled first strand and the extended labeled second strand of (iii).
  • a phosphate group preferably is not attached to 3' end of the oligonucleotide, which allows this end to be ligated to each strand of the fragment.
  • a phosphate group preferably is not attached to the 5' end of the oligonucleotide, which prevents this end from being ligated to the 3' end of the oligonucleotide, thereby preventing self-ligation of the oligonucleotide.
  • the 5' end of the oligonucleotide cannot be ligated to the 3' end of each of the first and second strands, which allows the latter end to be extended by a nucleic acid polymerase.
  • an end of the fragment is a blunt end
  • the 3' end of the first sequence and the 5' end of the third sequence advantageously form a blunt end in the oligonucleotide.
  • an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases)
  • the 3' end of the first sequence and the 5' end of the third sequence advantageously form a terminal overhang in the oligonucleotide that is complementary to the terminal overhang in the fragment.
  • the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
  • a linker-primer duplex which contains (i) a first oligonucleotide (a linker-primer/MET-labeled oligonucleotide), (ii) a second oligonucleotide (a ligation-aid/MET-labeled oligonucleotide), and (iii) a molecular energy transfer pair.
  • the molecular energy transfer pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first oligonucleotide is annealed to the second oligonucleotide.
  • the first oligonucleotide contains the donor moiety and the second oligonucleotide contains the acceptor moiety.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the linker-primer duplex is formed.
  • step (b) the 3' end of the first oligonucleotide is ligated to the 5' end of the first strand of the fragment to form a labeled first strand, and the 3' end of the first oligonucleotide is ligated to the 5' end of the second strand of the fragment to form a labeled second strand.
  • step (c) the 3' end of the labeled first strand is extended using the labeled second strand as a template to form an extended labeled first strand. Additionally, the 3' end of the labeled second strand is extended using the labeled first strand as a template to form an extended labeled second strand. Consequently, the extended labeled first strand is annealed to the extended labeled second strand.
  • step (d) which is optional, the extended labeled first and second strands are amplified.
  • step (e) which follows either step (b), (c), or (d)
  • the emitted energy e.g., light
  • step (d) the emitted energy (e.g., light) is detected in order to detect the fragment.
  • a variety of amplification methods can be used in step (d) to amplify the extended labeled first and second strands (e.g., PCR amplification, strand displacement amplification, and cascade rolling circle amplification).
  • the preferable method of amplification is PCR amplification comprising the following four steps:
  • step (i) the extended labeled first strand is separated from the extended labeled second strand.
  • step (ii) the first oligonucleotide is annealed to each of the extended labeled first and second strands.
  • step (iii) the 3' end of the first oligonucleotide is extended using the extended labeled first strand as a template to form another extended labeled second strand, wherein e extended labeled first strand is annealed to the other extended labeled second strand. Additionally, the 3' end of the first oligonucleotide is extended using the extended labeled second strand as a template to form another extended labeled first strand, wherein the extended labeled second strand is annealed to the other extended labeled first strand.
  • steps (i), (ii), and (iii) are repeated for a finite number of times, wherein, in (i), the extended labeled first and second strands respectively are the extended labeled first strand and the other extended labeled second strand of (iii), or respectively are the other extended labeled first strand and the extended labeled second strand of (iii).
  • a phosphate group preferably is not attached to the 3' end of the first oligonucleotide, which allows this end to be ligated to each strand of the fragment if each strand contains a phosphate group at the 5' end.
  • a phosphate group preferably is not attached to the 5' end of the second oligonucleotide, which prevents this end from being ligated to the 3' end of the first oligonucleotide, thereby preventing self-ligation of the linker-primer duplex.
  • the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands, which allows the latter end to be extended by a nucleic acid polymerase.
  • an end of the fragment is a blunt end
  • the 3' end of die first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a blunt end in the duplex.
  • an end of the fragment has a terminal overhang (e.g. , a terminal overhang of 1 to 10 bases)
  • the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide advantageously form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.
  • kits for detecting a double-stranded nucleic acid fragment comprises (a) ligase, (b) a first oligonucleotide, (c) a second oligonucleotide capable of annealing to the first oligonucleotide, and (d) a third oligonucleotide.
  • the third oligonucleotide contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair.
  • the molecular energy pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first nucleotide sequence is capable of annealing to the complement of the first oligonucleotide.
  • the second nucleotide sequence contains the donor moiety and the fourth nucleotide sequence contains the acceptor moiety, or the second nucleotide sequence contains the acceptor moiety and the fourth nucleotide sequence contains the donor moiety.
  • the second nucleotide sequence is capable of annealing to the fourth nucleotide sequence to form a hai ⁇ in.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the hai ⁇ in is formed.
  • a second kit comprises (a) ligase, (b) a first oligonucleotide, and (c) a second oligonucleotide.
  • the first oligonucleotide contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair.
  • the molecular energy pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
  • the first nucleotide sequence is capable of annealing to the second oligonucleotide.
  • the second nucleotide sequence contains the donor moiety and the fourth nucleotide sequence contains the acceptor moiety, or the second nucleotide sequence contains the acceptor moiety and the fourth nucleotide sequence contains the donor moiety.
  • the second nucleotide sequence is capable of annealing to the fourth nucleotide sequence to form a hai ⁇ in.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the hai ⁇ in is formed.
  • a third kit comprises (a) ligase, and (b) an oligonucleotide containing (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, and (iv) a molecular energy transfer pair.
  • the molecular energy transfer pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing d e emitted energy.
  • the first nucleotide sequence is capable of annealing to the third nucleotide sequence to form a hai ⁇ in.
  • the first nucleotide sequence contains the 3' end of the oligonucleotide.
  • the third nucleotide sequence contains the 5' end of the oligonucleotide.
  • the first nucleotide sequence contains the donor moiety and the third nucleotide sequence contains the acceptor moiety, or the first nucleotide sequence contains the acceptor moiety and d e third nucleotide sequence contains the donor moiety.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if die hai ⁇ in is formed.
  • a fourth kit comprises (a) ligase, (b) a first oligonucleotide, (c) a second oligonucleotide, and (d) a molecular energy transfer pair.
  • the molecular energy pair comprises an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing die emitted energy.
  • the first oligonucleotide is capable of annealing to the second oligonucleotide.
  • the first oligonucleotide contains die donor moiety and the second oligonucleotide contains the acceptor moiety.
  • the acceptor moiety absorbs a substantial amount of the emitted energy only if the first oligonucleotide is annealed to the second oligonucleotide.
  • Figure 1 is a schematic illustration of the FLM-PCR method of Examples 1 and 3 utilizing a linker-primer duplex and a detectable primer, wherein the linker-primer duplex contains an unlabeled linker-primer oligonucleotide and an unlabeled ligation-aid oligonucleotide, and me detectable primer is a FRET-labeled oligonucleotide containing a donor moiety (O) and an acceptor moiety (•);
  • Figure 2A is a schematic illustration of a reaction involving an unlabeled ligation- aid oligonucleotide and a FRET-labeled oligonucleotide containing a donor moiety (O) and an acceptor moiety (•), wherein the reaction potentially competes with FLM-PCR and increases background flourescence;
  • Figure 2B is a schematic illustration of a linker- primer duplex containing an unlabeled linker-primer oligonucleotide and an unlabeled ligation-aid oligonucleotide, wherein the ligation-aid oligonucleotide contains a modifier on its 3' end which blocks the potentially competing reaction illustrated in Figure 2A;
  • Figure 3 is a photograph of an agarose gel containing FLM-PCR reaction products obtained using different ligation-aid oligonucleotides
  • Figure 4 is a schematic illustration of the FLM-PCR method of Example 2 utilizing a linker-primer duplex, wherein the linker-primer duplex contains a FRET-labeled linker- primer oligonucleotide including a donor moiety (O) and an acceptor moiety (•), and an unlabeled ligation-aid oligonucleotide;
  • Figure 5A is a plot of fluorescence data obtained from FLM-PCR reactions, wherein different concentrations of thymus DNA were used; and Figure 5B is a calibration curve obtained by replotting data from 20 cycles of PCR amplification;
  • Figure 6 is a schematic illustration of a FLM-PCR mediod utilizing a FRET-labeled linker-primer oligonucleotide containing a donor moiety (O), an acceptor moiety (•), and a ligation-aid sequence
  • Figure 7 is a schematic illustration of a FLM-PCR method utilizing a linker-primer duplex, wherein the linker-primer duplex contains a FRET-labeled linker-primer oligonucleotide including a donor moiety (O), and a FRET-labeled ligation-aid oligonucleotide including an acceptor moiety (•); and
  • Figure 8 is a schematic illustration of the LM-PCR method of Staley et al. utilizing a linker-primer duplex containing an unlabeled linker-primer oligonucleotide and an unlabeled ligation-aid oligonucleotide.
  • Example 1 FLM-PCR Amplification and Detection Using One FRET-Labeled and Two Unlabeled Oligonucleotides Introduction: The FLM-PCR method of Example 1 is schematically illustrated in Figure 1.
  • DNA was purified from the thymus gland of adult male rats by adso ⁇ tion and elution using a Wizard MidiColumn affinity resin (Promega) according to Eldadah et al. (1996, Nucleic Acids Research 24:4092-4093, the entire contents of which are herein inco ⁇ orated by reference).
  • One rat was injected with dexameti sone to induce thymic regression. The rat subsequently was sacrificed after 20 hours. Tissues (60-89 mg) were homogenized in 200 ⁇ L of PBS using a loose-fitting plastic pestle in a microfuge tube.
  • DNA was eluted in 50 ⁇ L of TE buffer (Tris-HCl, 10 mM; EDTA, 1 mM) by centrifugation of die columns. DNA was measured by die PicoGreen Fluorescence Assay (Molecular Probes) using a Wallac 1420 spectrofluorometer. Purified DNA was stored at - 20°C until use.
  • TE buffer Tris-HCl, 10 mM; EDTA, 1 mM
  • oligonucleotides consisting of the following sequences were synthesized: 5'- TGCGGTGAACCT-(3'-C7-amine) (SEQ ID NO:7), 5'-TCGGGTGAACCTT-(3'-C3- phosphate) (SEQ ID NO:8), 5'-TCGGGTGAACCT-3'-OH (SEQ ID NO:9), 5'- CCTGCAGGCTGAGGT ⁇ CACCGCA-S' (SEQ ID NO: 10), and 5'-(fluorescein)- AGCTGGAACGCTATCCAGCT-(DABCYL -CCTGCAGGCTGAGGT-3 , (SEQ ID NO:ll).
  • SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 were used as ligation-aid oligonucleotides
  • SEQ ID NO: 10 was used as a linker-primer oligonucleotide
  • SEQ ID NO: 11 was used as a detectable FRET-labeled oligonucleotide.
  • SEQ ID NO:7 was synthesized using 3'-Amino-Modifier C7 CPG (Glen Research 20-2957)
  • SEQ ID NO: 8 was synthesized using 3'-Amino-Modifier C3 CPG (Glen Research 20-2950).
  • Each ligation reaction mixture contained one unlabeled ligation-aid oligonucleotide (SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9), and one unlabeled linker-primer (SEQ ID NO: 10).
  • Target DNA was ligated with the linker-primer oligonucleotide using a ligation-aid oligonucleotide as follows. Genomic DNA (1.0 ⁇ g) was mixed with SEQ ID NO: 10 and unphosphorylated SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 in 60 ⁇ L of T4 DNA ligase buffer (TaKaRa).
  • the ratio of genomic DNA to each oligonucleotide was 1 ⁇ g DNA per 0.07-1.0 nmole of each oligonucleotide. Oligonucleotides were annealed by heating each mixture in a PCR machine to 55 °C for 10 minutes, allowing the mixture to cool to 10°C over 55 minutes, incubating the mixture at 10°C for 10 minutes, and warming the mixture to 16°C. T4 DNA ligase (1.0 ⁇ L, Appligene, 1.5 Weiss U/ ⁇ L) was then added and mixed in the proportion of 1.5 units per ⁇ g DNA, and the reaction was continued for 16 hours at 16°C. The mixtures were then diluted with TE to a final concentration of 5 ng/ ⁇ L. Ligated DNA was stored at -20°C until use.
  • Ligase-mediated ligation is a very inefficient reaction when blunt ends are joined, because these ends do not anneal.
  • a high concentration of linker-primer oligonucleotide was used to drive the ligation reaction, which was also appropriate, considering that self- ligation of fragments of genomic DNA needed to be competitively blocked.
  • SEQ ID NO: 10 and an equal amount of ligation-aid oligonucleotide were both used in 10-fold excess over the potential maximum concentration of DNA ends in a reaction.
  • the potential concentration of DNA ends from a 100% apoptotic DNA sample is around 15 pmol of ⁇ 185 bp fragments/ ⁇ g DNA, and a working DNA concentration was 17 ng/ ⁇ L.
  • Each PCR reaction mixed from a ligation reaction mixture contained 10 pmol of one FRET-labeled oligonucleotide (SEQ ID NO: 11).
  • Ligated DNA (30 ng) was used in a PCR assay (20 ⁇ L volume reaction mixture) containing an additional 0 to 100 pmol of SEQ ID NO: 10, and 0 to 100 pmol of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, Taq Buffer (TaKaRa), supplementary MgCh (1 mM), and an equimolar mixture of 0.32 mM of each of dATP, dGTP, dCTP and dTTP. All reaction components were mixed on ice.
  • Taq polymerase was preincubated with TaqStart antibody, 1:1 (v:v) (ClonTech) for 10 minutes at 25 °C to inhibit Taq activity until the temperature reached 72°C.
  • Taq/TaqStart complex was added to reactions at 4°C.
  • a PCR tiiermal cycler widi a heated lid was programmed as follows: extension at 3' end of genomic DNA for 5-8 minutes; 12-40 PCR cycles consisting of 0 (pulse) to 60 seconds at 92 +/- 4°, and 0.5-5 minutes at 72° +/- 6°; and optionally 5 minutes at 72° to assure a completed final extension cycle.
  • the melting time usually used was 30 seconds and the extension time was 90 seconds per cycle.
  • the block of the thermal cycler was precooled to 4°C for 2 minutes, and then die samples were positioned in it.
  • Table 2 shows separate or conjoint effects of linear linker-primer oligonucleotide and linear ligation-aid oligonucleotide on background fluorescence in FLM-PCR in the presence of a FRET-labeled oligonucleotide.
  • the fluorescence intensity was determined after subtraction of background present before die PCR amplification.
  • FIG. 2A A model explaining the generation of this spurious fluorescence signal is shown in Figure 2A, which illustrates that a side reaction probably diverted a variable portion of d e FRET-labeled oligonucleotide, presumably by synthesizing a complementary strand.
  • a side reaction probably diverted a variable portion of d e FRET-labeled oligonucleotide, presumably by synthesizing a complementary strand.
  • its diversionary activity was inversely proportional to d e amount of genomic DNA. This diversionary activity caused a variable bias which interfered widi standardization and calibration of the PCR reaction.
  • Both the linker-primer oligonucleotide and die unmodified ligation-aid oligonucleotide were required to generate the spurious fluorescence signal.
  • Table 3 shows die effect of a 3' modification of a ligation-aid oligonucleotide on background fluorescence in FLM-PCR in the presence of a FRET-labeled hai ⁇ in primer oligonucleotide and in the absence of DNA.
  • the fluorescence intensity was determined after subtraction of background present before die PCR amplification.
  • Example 2 FLM-PCR Amplification Using One FRET-Labeled Oligonucleotide and One Unlabeled Oligonucleotide Introduction: The FLM-PCR method of Example 2 is schematically illustrated in Figure 4.
  • Example 2 demonstrates that the detectable FRET-labeled oligonucleotide used as a PCR primer in Example 1 (SEQ ID NO: 11) also can be used for direct ligation to a target DNA when this oligonucleotide is annealed to a ligation-aid oligonucleotide.
  • the reaction sequence of Example 2 was used to demonstrate a simple form of quantitative FLM-PCR analysis in which titrations of a standard are compared to samples to be analyzed by measuring direct fluorescence in die exponential amplification stage of the reactions. In this analysis, the percentage of adenine 3' overhanging ends could be directly compared to that of blunt ends.
  • a competitive non-labeled linker-primer oligonucleotide with a 5' degenerate sequence can be used to improve the specificity of the amplification reaction.
  • the linker- primer oligonucleotide must have a 3' sequence identical to about 2-10 bases at die 3' end of the linker-primer oligonucleotide, which is linked to a 5' sequence of about 10 random bases as described by Atamas et al., 1998, BioTechniques 24:445-450.
  • Thymus DNA was prepared as described in Example 1. This DNA was used as a reference DNA. DNA purified from rat liver or human placenta was tested against this reference DNA. Thymus DNA was also tested for adenine 3' overhanging ends.
  • the FRET-labeled oligonucleotide used in Example 1 (SEQ ID NO: 11) was used as a linker-primer oligonucleotide in Example 2. Additionally, two oligonucleotides consisting of the following sequences were synthesized: 5'-ACCTCAGCCTGC-C7-amine (SEQ ID NO: 12) and 5XCCTCAGCCTGCA-C7-amine (SEQ ID NO: 13).
  • SEQ ID NO: 11 was annealed to either SEQ ID NO: 12 or SEQ ID NO: 13.
  • the resulting linker-primer duplex containing SEQ ID NO: 11 and SEQ ID NO: 12 forms a blunt end.
  • the resulting linker-primer duplex containing SEQ ID NO: 11 and SEQ ID NO: 13 forms an overhanging deoxythymidine at the 3' end.
  • Concentrations of about 0.5 ⁇ M of each of SEQ ID NO: 11 and SEQ ID NO: 12 were used in the ligation step.
  • SEQ ID NO: 11 and either SEQ ID NO: 12 or SEQ ID NO:13 were annealed by heating to 55° and cooling to 10°.
  • SEQ ID NO: l l contains a 12- base sequence that is complementary to a 12-base sequence in each of SEQ ID NO: 12 and SEQ ID NO: 13. These complementary sequences anneal at about ⁇ 40° C but not at about ⁇ 40°C.
  • SEQ ID NO: 11 was then ligated to the genomic DNA at 16° C. The ligation reactions were done in total volumes that were scaled down to 5 ⁇ L, and reaction times were varied. Ligated samples were diluted about ten-fold into PCR reactions; upon dilution, d e concentration of SEQ ID NO: 11 was thus ⁇ 0.05 ⁇ M.
  • the PCR reaction mixtures contained 0.5 ⁇ M of SEQ ID NO: 11, Taq polymerase and reaction buffer (TaKaRa), dNTP mix (0.3 mM each of dTTP, dATP, dCTP & dGTP), and 0.19-1.5 ng DNA/ ⁇ l. Samples were reacted in a thermal cycler as follows: hold 1 for 5 min. at 72°; men 20 cycles of 0.5 min. at 94° and 1.5 min. at 72°. Fluorescence of fluorescein was measured in closed tubes in a Wallac spectrofluorimeter.
  • DNA was prepared from normal rat thymus using either a Promega Wizards Kit as described in Example 1, or using a Nonorganic DNA Extraction Kit (Oncor). Three sets of oligonucleotides were compared in me same reaction sequence. The first set of oligonucleotides was SEQ ID NO:7, SEQ ID NO: 10, and SEQ ID NO: 11. PCR controls contained either no DNA or no Taq polymerase.
  • oligonucleotides consisting of the following sequences: 5'-TGCGGTGAGAGTG-(3'-C7-amine) (SEQ ID NO: 14), S'-ACTGAACCTGACCGTACACTCTCACCGCA-S' (SEQ ID NO: 15),
  • the second set of oligonucleotides consisted of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. Identical sequence segments of SEQ ID NO: 15 and SEQ ID NO: 16 are underlined, and d e annealing relationships of SEQ ID NO: 14 and SEQ ID NO: 15 are illustrated in Figure 1, in which a 3' mark denotes a modified 3' end.
  • the third set of oligonucleotides consisted of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: l. Identical sequence segments of SEQ ID NO: 15 and SEQ ID NO: 1 are underlined
  • SEQ ID NO: 16 and SEQ ID NO:l each had a single-stranded stem that was less prone to primer-dimer formation tiian a single-stranded stem in SEQ ID NO: 11.
  • SEQ ID NO: 16 had a higher-melting self-annealing sequence than SEQ ID NO: 11, while SEQ ID No:l and SEQ ID NO: 11 had similar self-annealing sequences.
  • Ligation reactions and PCR reactions were performed similarly to the reactions Example 1. Results are given in Table 5. These data indicate that SEQ ID NO:l gave the highest ratio of sample/control fluorescence.
  • a preferred standard calibrator is one with a definite expected number of DNA ends, rather than an estimated number of ends.
  • a standard preferably should be ligated separately from genomic DNA. The efficiency of the ligation reaction of a standard should be the same as tiiat of the genomic DNA. This condition generally can be satisfied if d e standard and the genomic DNA are both ligated widi any complementary ended duplex having die same kind of ends. This specification normalizes out any effect of differential annealing of overhanging ends on the ligation reactions of standard or genomic DNA.
  • the sequence of d e linker-primer oligonucleotide tiiat is ligated to any kind of standard can be made different from the sequence of the linker-primer oligonucleotide tiiat is ligated to genomic DNA.
  • This difference confers the potential to use differently FRET-labeled oligonucleotides with different primer sequences in die amplification of the standard DNA and the genomic DNA.
  • Ligated standard can be mixed with genomic DNA in one amplification reaction, thus allowing for the possibility of multiplex amplification and detection of botii kinds of amplicons at the same time. Standards for two different uses are described.
  • the invention relates to a comparison of data obtained from eitiier a parallel or a multiplex reaction comprising one or more standards.
  • the standard calibrator and its ancillary oligonucleotides preferably may be included in a kit of die invention.
  • a sample of DNA purified from apoptotic cultured cells is one kind of a standard which may be used for calibrating the relative number of DNA ends.
  • a standard is likely to contain a very wide range of DNA fragment sizes, each of which will be amplified widi a different efficiency.
  • the efficiency is usually inversely proportional to the size. The number of ends in it cannot be exactly calculated.
  • the preferred kind of standard calibrator for enumerating DNA ends is a set of a small number of DNA fragments having a narrow range of sizes spanning die range of about 150-2000 bp, and having only one specific kind of end.
  • DNA fragments can be prepared by reacting a bacterial duplex DNA plasmid with a pure restriction endonuclease. This process can give a defined number of ends per ⁇ g DNA when reacted to completion, if me plasmid and die nuclease pair is carefully selected from a group for which their reaction is well understood.
  • the present invention can be varied to detect blunt DNA ends, or slightly overhanging 3' or 5' ends, or multiple kinds of ends in one test.
  • the best sensitivity can be obtained by detecting multiple kinds of ends, but by this me od, it may be difficult to provide a single standard calibrator comprising all equivalent ends.
  • die highest quality can be obtained using commercially available restriction enzymes if those forming blunt-ended fragments are selected.
  • Calibrator 1 comprised blunt-ended DNA fragments made by cutting d e plasmid SV40 with die endonuclease Ssp I, from which six fragments were expected (153 bp, 529 bp, 803 bp, 871 bp, 1433 bp, and 1463 bp). Enzyme, plasmid, and buffers were purchased from GIBCO-BRL.
  • Calibrator 2 comprised blunt-ended DNA fragments made by cutting die plasmid LITMUS 28 widi the endonuclease Mspl I, from which four fragments were expected (292 bp, 1724 bp, 1969 bp and 2708 bp).
  • Enzyme, plasmid, and buffer were purchased from New England Biolabs.
  • Calibrator 3 comprised fragments with a one-base 3' overhanging end made by cutting die plasmid pBR322 with the endonuclease Bmr I, from which five fragments were expected (69 bp, 306 bp, 1192 bp, 1254 bp, and 1540 bp).
  • 1 or 2 ⁇ g of plasmid was diluted in 10 ⁇ l nuclease specific buffer containing 0-6 units of endonuclease, and these were incubated for one hour at 37°. Enzymes were then denatured at 65° for 20 min. Agarose gels (1.3%) were run in TBE buffer.
  • the blunt- ended fragments of both kinds used were ligated to SEQ ID NO: 15, in buffer with 1.0 mM or 0.05 mM ATP, for 16 hours at 16°, and frozen until use. They were diluted into PCR reactions with SEQ ID NO:l, 0.5 ⁇ M.
  • the present invention includes a "multiplex PCR" mediod utilizing first and second linker-primer oligonucleotides, wherein the first linker-primer oligonucleotide is labeled with a first MET pair (e.g., fluorescein and DABCYL), and the second oligonucleotide is labeled with a second MET pair, e.g., Texas Red (or rhodamine) and DABCYL tiiat is different from die first MET pair.
  • a first MET pair e.g., fluorescein and DABCYL
  • second MET pair e.g., Texas Red (or rhodamine)
  • DABCYL tiiat is different from die first MET pair.
  • Any two multiplexed reactions should be performed using oligonucleotides of nearly identical secondary structures and physicochemical characteristics, differing only in dieir MET labels and oligonucleotide sequences. Multiplexation confers the advantage of identical reaction conditions and measurement conditions for die two reactions, significantly increasing the accuracy of measurement. Because PCR is a method for geometric amplification, measurable differences can arise between samples due to even small differences in tiieir internal conditions during thermal cycling.
  • a very accurate mediod of calibration involves ligation of a portion of a standard calibrator wid an oligonucleotide differing sufficiently in sequence from the oligonucleotide tiiat is ligated to the genomic DNA sample.
  • Different MET-labeled PCR primers are provided for the internal control reaction and the genomic DNA reaction, which have been qualified so as not to cross-anneal. Multiplex PCR is performed, and two fluorescence channels are used for measurment.
  • the present invention includes a method to provide an internal standard with which the number of genomic DNA ends can be compared.
  • a first MET-labeled primer, and any ancillary strands depending on the selected embodiment, are used to measure genomic DNA ends.
  • the internal calibrator comprises two parts. One part is a second, MET-labeled primer having both a different color and a different nucleotide sequence than the first.
  • the second part is a linker-primer strand that comprises a sequence to which the second MET-labeled primer can anneal at the 3' end, which is different dian the sequence to which die first MET-labeled primer can anneal. If the procedure is done using the multiplex mode, appropriate normalization calculations must be done to account for differences in intrinsic brightness and background signal.
  • Another use for a standard serving as a control is to assure that die relative amount of DNA per sample is consistent and reproducible.
  • a simple method for estimation of the relevant statistical measures is to amplify a specific gene sequence in a parallel reaction.
  • a better, more informative method is to amplifying the control gene sequence in the same reaction mixtures as the analyte, using multiplex amplification and detection.
  • the multiplex method requires the use of anotiier pair of MET labeled primers that have been optimized to amplify under die same reaction conditions as the genomic DNA.
  • Two examples of genes at are known in the art to be amenable for this use are the glyceraldehyde-3-phosphate dehydrogenase gene and the actin gene.
  • One set of oligonucleotides is SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:l.
  • a second set of oligonucleotides is chosen to have die same lengtii, d e same annealing temperature, and similar percentages of purine and pyrimidine bases as d e first set.
  • the analog of SEQ ID NO: 1 is labeled with a different fluorophore and quencher, such as rhodamine and DABCYL. This similarity allows one to do PCR amplification of bom sets under very similar conditions to those which are used for the reaction without the internal standard. The amount of internal standard is titrated in several replicates of different samples of me genomic DNA.
  • the titration of internal standard provides one or more reactions in which the fluorescence of standard and genomic sample are similar, relative to their respective dynamic ranges and backgrounds. If d e measurements are within a statistically validated zone of the assay, die proportionality of the sample to the standard allows for doing a measurement of the amount of sample ends.

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Abstract

L'invention concerne des méthodes d'amplification en chaîne par polymérase induite par ligase (LM-PCR) utilisées pour détecter des fragments cibles d'acides nucléiques à double brin tels que ceux qui sont générés lors du processus apoptosique. Selon ces méthodes, un oligonucléotide détectable est incorporé dans la cible. Cet oligonucléotide détectable renferme la fraction donneur et/ou la fraction receveur d'une paire de transfert d'énergie moléculaire (MET). Un exemple de paire MET est une paire de transfert par résonance d'énergie de fluorescence (FRET) constituée d'une fraction fluorophore (donneur) et d'une fraction extinctrice de luminescence (receveur). La fraction donneur de la paire MET émet de l'énergie détectable telle que de la lumière seulement lorsque l'oligonucléotide détectable est incorporé dans la cible. Selon ces méthodes, un oligonucléotide lieur-amorce est attaché à un oligonucléotide d'aide à la liaison, ou un oligonucléotide lieur-amorce renfermant une séquence d'aide à la liaison est lié à l'extrémité 5' de chacun des brins d'un fragment d'acides nucléiques à double brin renfermant soit une extrémité franche soit un surplomb terminal. Après cette étape de liaison, un oligonucléotide détectable capable d'être attaché au complément de l'oligonuléotide lieur-amorce est incorporé dans la cible par réactions catalysées par polymérase. Selon un autre mode de réalisation, l'oligonucléotide lieur-amorce est également un oligonucléotide détectable. Eventuellement, la cible marquée par l'oligonucléotide détectable est ensuite amplifiée, l'oligonucléotide détectable étant incorporé au produit d'amplification. La cible est détectée par détection de l'énergie émise par le groupe donneur de l'oligonucléotide détectable.
PCT/US1998/026432 1997-12-12 1998-12-11 Methode de detection de l'apoptose a l'aide d'oligonucleotides marques par fret WO1999029905A2 (fr)

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US10131789B2 (en) 2015-03-30 2018-11-20 Enzo Biochem, Inc. Monoazo dyes with cyclic amine as fluorescence quencers
US10865309B2 (en) 2015-03-30 2020-12-15 Enzo Biochem, Inc. Monoazo dyes with cyclic amine as fluorescence quenchers
EP4008790A1 (fr) 2015-03-30 2022-06-08 Enzo Biochem, Inc. Colorants monoazoïques dotés d'une amine cyclique en tant qu'extincteurs de fluorescence
WO2016160051A1 (fr) 2015-03-30 2016-10-06 Enzo Biochem, Inc. Colorants monoazoïques dotés d'une amine cyclique en tant qu'extincteurs de fluorescence
CN109182479A (zh) * 2018-07-27 2019-01-11 广州医科大学附属第医院 一种实时荧光定量pcr通用型荧光探针及其应用

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EP1036201A2 (fr) 2000-09-20
WO1999029905B1 (fr) 1999-10-21

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