CA2314225A1

CA2314225A1 - Method for detecting apoptosis using fret labeled oligonucleotides

Info

Publication number: CA2314225A1
Application number: CA002314225A
Authority: CA
Inventors: William M. James; Irina A. Nazarenko
Original assignee: Intergen Co
Current assignee: Individual
Priority date: 1997-12-12
Filing date: 1998-12-11
Publication date: 1999-06-17
Also published as: WO1999029905A3; JP2001526052A; WO1999029905B1; EP1036201A2; WO1999029905A2

Abstract

Ligase-mediated polymerase chain reaction (LM-PCR) methods are used to detect target double-stranded nucleic acid fragments such as those generated during the process of apoptosis. In these methods, a detectable oligonucleotide is incorporated into the target. This detectable oligonucleotide contains the donor moiety and/or the acceptor moiety of a molecular energy transfer (MET) pair. An example of a MET pair is a fluorescence energy resonance transfer (FRET) pair consisting of a fluorophore (donor) moiety and a quencher (acceptor) moiety. The donor moiety of the MET pair emits detectable energy such as light only when the detectable oligonucleotide is incorporated into the target. In these methods, a linker-primer oligonucleotide annealed to a ligation-aid oligonucleotide, or a linker-primer oligonucleotide containing a ligation-aid sequence, is ligated to the 5' end of each strand of a doublestranded nucleic acid fragment containing either a blunt end or a terminal overhang. After this ligation step, a detectable oligonucleotide capable of annealing to the complement of the linker-primer oligonucleotide is incorporated into the target by polymerase-catalyzed reactions. Alternatively, the linker-primer oligonucleotide is also a detectable oligonucleotide. Optionally, the target labeled by the detectable oligonucleotide is subsequently amplified, wherein the detectable oligonucleotide is incorporated into the amplification product. The target is detected by detecting the energy emitted by the donor moiety of the detectable oligonucleotide.

Description

METHOD FOR DETECTING APOPTOSIS USING
FRET LABELED OLIGONUCLEOTIDES
BACKGROUND OF THE INVENTION
Field of the Invention:
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 polymerise chain reaction (LM-PCR) methods in which an aligonucleoude 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.
Description of the Related Art:
All muldcellular organisms continuously delete and replace some of their cells.
Cells are selectively deleted by a process referred to as apoptosis (also called cellular suicide or programmed cell death). Normally, 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.
During apoptosis, nucleic acids are hydrolyzed by cellular nucleases. However, in the cell, 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 nucleosames 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. This ladder, in which the fragment lengths are integral multiples of fram 180 to 200 bp, is widely regarded as a biochemical hallmark of apoptosis.

At any given time, 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 quandtate 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 &
Di,,~''erentiadon 4:66-7~, describe a method for detecting these DNA ends.
Specifically, Staley et al. adapted the method of LM-PCR for the amplification of apoptotic nucleosomal DNA fragments (see Figure 8).. In the method of Staley et al., the amplified DNA
fragments are quantitated using a gel electrophoretic assay in which sample band densities are compared. However, the gel electrophoresis assay of Staley et al. is labor-and time-intensive. Acxordingly, there is a need in the art for improved methods of detecting DNA
fragments resulting from the process of apoptosis.
SUMMARY OF THE INVENTION
The present invention is directed to LM-PCR methods for detecting DNA
fragments resulting from the process of apoptosis. In these methods, a labei is incorporated directly into the amplification product, making the amplification product immediately detectable without opening the reaction vessel. These methods therefore are superior to the method of Staley et al.
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 accxptor 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. In FRET, energy is passed between a danor molecule, which is a fluorophore, and an acceptor molecule.
The donor molecule absorbs a photon and transfers this energy to the acceptor molecule.
FBrster, 1949, Z. Naturforsch A4: 321-327; Clegg, 1992, 211 METHODS IN
ENZYMOLOGY 353-388. Pairs of molecules that can engage in MET or FRET are termed MET pairs or FRET pairs, respectively.

When two fluorophores whose excitation and emission spectra overlap are in close proximity, 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. As a result, the fluorescence of the donor molecule is quenched, while the fluorescence of the acceptor molecule is enhanced. When the excited-state energy of the donor is transferred to a non-fluorophore acceptor, the fluorestxnce of the donor is quenched without subsequent emission of fluorescence by the acceptor. 1n 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 oligonucleodde." 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. 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 accxptor 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.
When an oligonucleotide containing a donor moiety is incorporated into an amplification product, 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. Ttus 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).

In one embodiment of the invention, illustrated schematically in Figure 1, the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
In step (a), a linker-primer duplex is provided which contains a first oligonucleotide (a linker-primer oligonucleotide) and sevond oligonucleotide (a ligation-aid oligonucleotide), wherein the first oligonucleotide is annealed to the second oligonucleotide. Also provided is 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. 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 nucieoti~ sequence contains the donor moiety.
The second nucleotide sequence is annealed to the fourth nucleotide sequence to form a hairpin. The acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.
In step (b), the 3' end of the first oligonucleotide is ligaied 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 S' end of the second strand of the fragment to form a ligated second strand.
In 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.
In step (d), the extended first strand is separated from the extended second strand.
In step (e), the third oligonucleodde is annealed to each of the extended first and second strands.

In 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 tempiate to form a doubly extended first strand. Additionally, the 3' end of the third oligonucleo'de is extended using the extended second strand as a template to form a labeled first strand, and 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.
In 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.
In step (h), the third oligonucleotide is annealed to each of the labeled first and second strands, and to each of the ~ubly extended first and second strands.
In step (i), the 3' end of the third oligonucieotide is extended using the labeled first strand and the d~bly 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.
In step (j), which is optional, the extended labeled first and second strands are amplified.
In step (k), which follows either step (fj, (i), or (j), the emitted energy (e.g., light) is detected in order to detect the fragment.
Those of ordinary skill, in the art understand that 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:
In step (i), the extended labeled first strand is separated from the extended labeled second strand.
In step (ii), the third oligonucleotide is annealed to each of the extended labeled first and second strands.
In step (iii), the 3' end of the third oligonucleotide is extended using the extended labeled first strand ax 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.
In step (iv), 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).
Advantageously, the donor moiety is a fluorophore and the accxptor moiety is a quencher of light emitted by the fluorophore. The donor moiety may be fluorescein, 5-carboxyfluorescxin (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.
Preferably, the donor moiety is fluorescein, and the acceptor moiety is DABCYL, and the donor and acceptor moieties are respectively attached to complementary first and second nucleotides in the hairpin 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. Additionally, a phosphate group preferably is not attached to the 3' end of the first oligonucleodde, which allows this end to be ligated to each strand of the fragment if each strand contains a phosphate group at the 5' end. Also, 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. Preferably, 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 polymerise.
Preferably, the third oligonucleotide consists of the sequence 5'-AGCTGGAACTCTATCCAGC,TACTGAACCTGACCGTACA-3' (SEQ ID NO:1), 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 N0:2), 5'-ATGTACGGTCAGCi-3' (SEQ ID N0:3), 5'-TTGTACGGTCAGG-3' (SEQ ID N0:4), 5'-CTGTACGGTCAGG-3' (SEQ ID NO:S), and 5'-GTGTACGGTCAGG-3' (SEQ ID
N0:6).
If an end of the fragment is a blunt end, then the 3' end of the first oligonucleoti~
and the 5' end of the second oligonucleotide advantageously form a blunt end in the duplex. Alternatively, if an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases), then 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.
In a second embodiment of the invention, illustrated schematically in Figure 4, the method for detecting a. double-stranded nucleic acid fragment comprises the following steps:
In step (a), a linker-primer duplex containing a first oligonucleodde (a linker-primer/MET-labeled oligonucleodde) 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 hairpin. The acxeptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.
In 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.
In 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.
In step (d), which is optional, the extended labeled first and second strands are amplified.
In step (e), which follows step (b), (c), or (d), the emitted energy (e.g., light) is detected in order to detect the fragment.
Those of ordinary skill in the art understand that 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 displacxment 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.
In step (ii), the first oligonucleotide is annealed to each of the extended labeled first and second strands.
In 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 oligonucleodde 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.
In step (iv), 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. Also, a phosphate group preferably is not attached to the 5' end of the second oligonucleodde, which prevents this end from being ligated to the 3' end of the first oligonucleotide, thereby preventing self ligation of the linker-primer duplex. Preferably, the 5' end of the second oligonucleodde 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 polymerise.
_g_ 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. Alternatively, if an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases), then 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.
In a third embodiment of the invention, illustrated schematically in Figure 6, the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
In step (a), a linker-primer oligonucleotide (which also acts as a ligation-aid/MIrT-iab~eled 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 nucleatide sequence is annealed to the third nucleotide sequence to form a hairpin. 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 hairpin is formed.
In 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.
In 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 suand 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.
_g_ WO 99/29905 PCT/US9$/Z643Z
In step (d), which is optional, the extended labeled first and second strands are amplified.
In step (e), which follows step (b), (c), or (d), the emitted energy (e.g., light) is detected in order to detect the fragment.
'those of ordinary skill in the art understand that 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.
In step (ii), the oligonucleotide is annealed to each of the extended labeled first and send strands.
In 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.
In step (iv), sups (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. Also, 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. Preferably, the S' 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.
If an end of the fragment is a blunt end, then the 3' end of the first sequence and the 5' end of the third sequence advantageously form a blunt end in the oligonucleotide.
Alternatively, if an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases), then 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.
In a fourth embodiment of the invention, illustrated schematically in Figure 7, the method for detecting a double-stranded nucleic acid fragment comprises the following steps:
In atep (a), a linker-primer duplex is provided which contains (i) a first oligonucleotide (a linker-primer/MET-labeled oligonucleotide), (ii) a second oligonucleodde (a ligation-aid/ME'T-labeled oligonucleotide), and (iii) a molecular energy transfer pair. The molecular energy transfer pair comprises an energy donor moiety cable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy.
The first oligonucieotide is annealed to the second oligonucleotide. The first oligonucleotide contains the donor moiety and the second oligonucleodde contains the acceptor moiety. The acceptor moiety absorbs a substantial amount of the emitted energy only if the linker-primer duplex is formed.
In 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.
In step (c), the. 3' end of the labeled first strand is extended using the labeled second strand as a template tn 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.
In step (d), which is optional, the extended labeled first and second strands are amplified.
In step (e), which follaws either step (b), (c), or (d), the emitted energy (e.g., light) is detected in order to detect the fragment.
Those of ordinary skill in the art understand that 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.
In step (ii), the first oiigonucleodde is annealed to each of the extended labeled first and second strands.
In 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.
In step (iv), 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 grip preferably is not attached to the 3' end of the first oligonucieotide, which allows this end to be ligated to each strand of the fragment if each strand contains a phosphate graup 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. Preferably, the 5' end of the second oligonueleotide 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 polymerise.
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. Alternatively, if an end of the fragment has a terminal overhang (e.g., a terminal overhang of 1 to 10 bases), then 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 present invention also relates to kits for detecting a double-stranded nucleic acid fragment. One kit 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 oligonucieotide 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. 'fhe 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 sevond nucleotide sequence is capable of annealing to the fourth nucleotide sequence to form a hairpin. The acxeptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.
A second kit comprises (a) ligase, (b) a first oligonucleotide, and (c) a second oligonucleodde. The first oliganucleodde 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. ',Che molecular energy pair comprises an energy donor moiety capable of emitting energy, and an energy acxepcor moiety capable of absorbing the emitted energy.
The first nucleotide sequence is capable of annealing to the second oligonucleodde.
The second nucleotide sequence contains the donor moiety and the fourth nucleotide ssquence 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 hairpin. The acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin 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 the emitted energy.
The first nucleotide sequence is capable of annealing to the third nucleotide sequence to form a hairpin. The first nucleotide sequence contains the 3' end of the S oligonucleodde. 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 acxeptor moiety, or the first nucleotide sequence contains the acxeptor moiety and the third nucleotide sequence contains the donor moiety. The acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin 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 the emitted energy.
The first oligonucleotide is capable of annealing to the second oligonucleodde. 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 first oIigonucleotide is annealed to the second oligonucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention may be understood more fully by reference to the following drawings:
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 the detectable primer is a FRET-Labeled oligonucleotide containing a donor moiety (O) and an aaxptor 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; and Figure 2B is a schematic illustration of a linker-primer duplex containing an unlabeled linker-primer oligonucleotide and an unlabeled WO 99/29905 PCf/US98/26432 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 SA is a plot of fluorescence data obtained from FLM-PCR reactions, wherein different concentrations of thymus DNA were used; and Figure SB is a calibration curve obtained by replotting data from 20 cycles of PCR amplification;
Figure 6 is a schematic illustration of a FLM-PCR method 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 oligonucleodde 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 tinker-primer oligonucleotide and an unlabeled ligation-aid oligonucleotide.
DETAILED DESCRIPTION OF THE INVENTION
Examples The Examples below are set forth by illustration only, and nothing therein shall be taken as a limitation upon the overall scope of the invention.

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.
Materials and Methods:
DNA was purified from the thymus gland of adult male rats by adsorption 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 incorporated by reference). One rat was injected with dexamethasone 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. 1.0 mL of 7.0 M guanidine HCI was added and mixed, and cellular debris was peileted by centrifugation at 14,000 x g for 15 minutes. Supernatant was mixed with 3.0 mL
of affinity matrix (Wizard Midipreps Purification Resin, Promega); the resulting mixture was packed in a Wizard Midicolumn under vacuum filtration. Columns were washed with potassium acetate, 8 mM; Tris-HCI, pH 7.5, 8.3 mM; EDTA, 0.04 mM; ethanol, 55.3 %
v:v. DNA was eluted in 50 ~L, of TE buffer ('iris-HCI, 10 mM; EDTA, 1 mM) by centrifugation of the columns. DNA was measured by the PicoGreen Fluorescence Assay (Molecular Probes) using a Wallac 1420 spectrofluorometer. Purified DNA was stored at -20°C until use.
Five oligonucleotides consisting of the following sequences were synthesized:
5'-TGCGGTGAACCT-(3'-C7-amine) (SEQ ID N0:7), 5'-TCGGGTGAACCTT-(3'-C3-phosphate) (SEQ ID N0:8), 5'-TCGGGTGAACCT-3'-OH (SEQ ID N0:9), 5'-CCTGCAGGCTGAGGTTCACCGCA-3' (SEQ ID N0:10), and 5'-(fluoresctin)-AGCTGGAACGCTA'TCCAGCT-{DABCYL)-CCTGCAGGCTGAGGT-3' {SEQ ID
NO:11). SEQ ID N0:7, SEQ ID N0:8, and SEQ ID N0:9 were used as ligation-aid oligonucleotides, SEQ ID N0:10 was used as a linker-primer oiigonucleotide, and SEQ ID
NO:11 was used as a detectable FRET-labeled oligonucleotide. SEQ ID N0:7 was synthesized using 3'-Amino-Modifier C7 CPG (Glen Research 20-2957), and SEQ ID
N0:8 was synthesized using 3'-Amino-Modifier C3 CPG (Glen Research 20-2950).
Identical sequence segments of SEQ ID NO:10 and SEQ ID NO:11 are underlined, and the annealing relationships of SEQ ID N0:7, SEQ ID N0:8, and SEQ ID N0:9 with SEQ
ID
NO:10 are illustrated in Figure 1, in which a 3' mark denotes a modified 3' end.
Each ligation reaction mixture contained one unlabeled ligation-aid oligonucleotide (SEQ ID N0:7, SEQ ID N0:8, or SEQ ID N0:9), 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 ~cg) was mixed with SEQ ID
NO:10 and unphosphorylated SEQ ID N0:7, SEQ ID N0:8, or SEQ ID N0:9 in 60 uL
of T4 DNA ligase buffer (TaKsRa). The ratio of genomic DNA to each oligonucleotide was 1 Pg DNA per 0.07-1.0 nmole of each oligonucleotide. Oligonucleotides were annealed by IO 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 ~cg 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/p,L. Ligated DNA was stored at -20°C until use.
Ligase-mediated ligation is a very inefficient reaction when biunt 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. For the most complete reaction, SEQ ID N0:10 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 by 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 N0:11). 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 11~ pmol of SEQ ID N0:7, SEQ ID N0:8, or SEQ ID N0:9, Taq Buffer (TaKaRa), supplementary MgClx (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.
In some samples, Taq polymerise was preincubated with TaqStart antibody, 1:1 (v:v) (CIonTech) 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 thermal cycler with a heated lid was programmed as follows: extension at 3' end of genomic DNA l:or 5-8 minutes; 12-40 PCR cycles consisting of 0 (pulse) to 60 seconds at 92 +/ 4°, and 0.5-~ 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 dme was 90 seconds per cycle. The block of the thermal cycler was precooled to 4°C for 2 minutes, and then the samples were positioned in it.
To obtain data in Figure 3, some tubes identified in this figure were placed in the thermal cycler block at 4° and rapidly camped to 72°C; some tubes were placed in the block at 25°C, and some tubes were placed in the block at 72°C.
The temperature was changed to 72°C for 8 minutes, during which the 5' protruding ends of the linker oligonucleotides were .filled in. PCR thermal cycles were done at 94°C
for 1 minute and 72°C for 5 minutes, for 20 cycles. A final step at 72°C for 5 minutes was used to assure complete synthesis in the last cycle. Ammonium sulfate at a concentration of 5-15 mM, was found to have an inhibitory effect on the post-ligation PCR reaction.
Samples in unopened PCR tubes were put into a 96-tube rack, and fluorescence was measured for one second in a Wallace Victor 1420 Multilabel Counter. Some samples were opened, diluted 1:100 into measuring buffer (0.002 M MgCls, 0.15 M NaCI, 0.01 M
Tris, pH 7.4), and read in a cuvette in a Shimadzu RFSOOOU speotrofluorimeter to compare results. Additionally, some samples were then analyzed by electrophoresis through a 1.2% agarose gel to examine the banding of the nucleosomal DNA
ladder.
Results:
When data on fluorescence intensity were compared with data on DNA fragment lengths as seen in gels., evidence of a side-reaction was observed. PCR
reactions containing linker-primer duplex, but either without genomic DNA or with unligated DNA, gave a high blank signal. As visualized by fluorescein fluorescence in agarose gels, fluorescence that was not associated with the DNA ladder appeared as a < 100 by smear.
After PCR
amplification, the fluorescence intensifies of the reaction mixture were measured, and these data are shown in Table 1.
Table 1 shows the effect of the concentration of the linker-primer duplex (containing a linker-primer oligonucleotide and an unmodified ligation-aid oligonucleotide) on the background fluorescence in the FLM-PCR method in which a FRET-labeled oligonucleotide and an unmodified ligation-aid oligonucleotide were used with no genomic DNA. The fluorescence intensity was determined after subtraction of background present before the PCR amplification.
Table 1 Concentration of + Taq (counts/20 ~cL)- Taq (counts/20 ~L) linker- rimer du lex (~M) 0.0 116,980 n/d 0.5 143,420 85,957 1.5 225,129 88,508 5.0 442,484 87,905 This background was doubled when both the linker-primer oligonucleotide and the unmodified ligation-aid oligonucleotide were reacted with the FRET-labeled oligonucleotide, as compared to either of them reacted separately, suggesting that both were involved in its cause (see Table 2).
Table 2 shows separate or conjoint effects of linear linker-primer oligonucleotide and linear ligadon-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 the PCR amplification.
Table 2 Concentration of 'linker-primerConcentration oligonucleotideof FRET-oligonucleotide~~or labeled PCR cycled fluorescence ligadon-aid oligonucleotide oligonucleodde(pM) M

SE ID NO:10 1.0 0.5 es 58,137 SE ID N0:9 1.0 0.5 es 52,856 both, each 1.0 0.5 s 105,576 neither 0 0.5 es 56,446 neither 0 0.5 no 51,515 both, each 1.0 0.5 no 49,534 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 the FRET-labeled oligonucleotide, presumably by synthesizing a complementary strand. As a competitive PCR reaction, its diversionary activity was inversely proportional to the amount of genomic DNA. This diversionary activity caused a variable bias which interfered with standardization and calibratian of the PCR reaction. Both the linker-primer oligonucleotide and the unmodified ligation-aid oligonucleotide were required to generate the spurious fluorescence signal.
A solution to this problem was found by modifying the 3' end of SEQ ID N0:9 with a suitable chemical modifier for blacking Taq polymerise {see Figure 2B).
Two modifiers were tested: 3'-Amino-Modifier C7CPG and 3'-Spacer C3 CPG {Glen Research). While the unmodified ligation-aid oligonucleotide gave a background proportional w the concentration used, the blocked ligation-aid oligonucleotides did not (see Table 3). Though observed fluorescence counts suggested that both modifiers effectively suppressed the incremental background, a gel electrophoretic assay showed that no PCR reaction products formed when the 3'-Spacer C3 CPG was used. By blocking the side-reaction with 3'-Amino-Modifier, the reaction was thus rendered amenable to calibration and standardization.
Table 3 shows the effect of a 3' modification of a ligadon-aid oligonucleotide on background fluorescence in FL,M-PCR in the presence of a FRET-labeled hairpin primer oligonucleotide and in the absence of DNA. The fluorescence intensity was determined after subtraction of background present before the PCR amplification.
Table 3 Concentration of ligation-aid oligonucleotide C3P C7N CH
in PCR M) 0.66 20,624 14,947 34,594 0.33 18,914 16,523 24,173 0 17,251 16,371 18,792 Those skilled in the art understand that it is possible to block extension of the 3' terminus of the ligation-aid oligonucleotide using a dideoxynucleotide. Liao et al., 1997, Analytical Biochemistry 253:137-139.

WO 99/29905 PCTlUS98rI6432 Example 2 FLM-PCR Amplification Using One FRET-Labeled Oliaonucleotide and One Unlabeled Oli~onucleotide 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 ligadon-aid oligonucleotide. The reaction sequence of Example 2: was used to demonstrate a simple form of quantitative FLM-PCR
analysis in which titcations of a standard are compared to samples to be analyzed by measuring direct fluorescence in the exponential amplification stage of the reactions. In this analysis, the percxntage 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 the 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, BioTechniquts 24:445-450.
Materials and Methods:
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 oligonucieotide used in Example 1 (SEQ ID NO:11) was used as a linker-primer oligonucleodde in Example 2. Additionally, two oligonucieotides consisting of the following sequences were synthesized: S'-ACCTCAGCGTGC-C7-amine (SEQ ID N0:12) and ;S'-CC;TC:AGCCTGCA-C7-amine (SEQ ID N0:13).
Before ligation, SEQ IT) NO:11 was annealed to either SEQ ID N0:12 or SEQ ID
N0:13. The resulting Linker-primer duplex containing SEQ ID NO:11 and SEQ ID
N0:12 forms a blunt end. The resulting linker-primer duplex containing SEQ ID NO:11 and SEQ
ID N0:13 forms an overhanging deoxythymidine at the 3' end.
Concentrations of about 0.5 ~M of each of SEQ ID NO:11 and SEQ ID N0:12 were used in the ligation step. SEQ ID NO:11 and either SEQ ID N0:12 or SEQ ID

N0:13 were annealed by heating to 55° and cooling to 10°. SEQ ID
NO:11 contains a 12-base sequence that is complementary to a 12-base sequence in each of SEQ ID
N0:12 and SEQ ID N0:13. These complementary sequences anneal at about 5 40° C but not at about z 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 ~cL, and reaction times were varied. Li,gated samples were diluted about ten-fold into PCR
reactions; upon dilution, the concentration of SEQ ID NO:11 was thus 50.05 ~cM.
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 DNAA~I. Samples were reacted in a thermal cycler as follows:
hold 1 for 5 min. at 72°; then 20 cycles of 0.5 min. at 94° and i.5 min. at 72°. Fluorescence of fluorescein was measured in closed tubes in a Wallac spectrofluorimeter.
Results:
The reaction kinetics found using FLM-PCR with different concentrations of thymus DNA fragments are as shown in Figure SA. The exponential phase of amplification under the chosen conditions extended from about cycles 18 to 30. By replotting some of these data at 20 cycles only, as shown in Figure 5B, a calibration curve was generated for comparison of other samples. Fluorescence measurements from samples were interpolated graphically to calculate the concentration of the kinds of DNA ends measured according to the end of linker-primer duplex. The comparison is shown in Table 4.
In Table 4, direct fluorescence was measured after 20 PCR cycles. In samples containing genomic D;NA, the concentration of the genomic DNA was 0.75 ng/~cl.
The column labeled "equiv./actuat x100°&" indicates a comparison between different sample DNAs and blunt ends measured in a "reference DNA" from dexamethasone-treated rat thymus. This result reflects the amount of ends measured in sample DNAs expressed as a percentage of the amount of blunt ends measured in "reference DNA." Normal liver and placenta DNAs have, as expected, much lower levels of blunt ends than has thymus after induction. The number of 3' overhanging dA ends measured in the same induced rat thymus was about one third that of blunt ends, assuming that these were ligated with equal efficiency. The reaction sequence of Example 2 gave a relatively high blank as tested with these particular oligonucleotide sequences.

Table 4 fluores. equiv.
DNA in uiv./

sample DNA fluores. units s~~s thymus ( ~~ ends units minus DNA
) blank n L) liver 0.75 blunt. 14 386 3,102 sam le 0.13 17.6 lacenta 0.75 blunt. 13,421 2,137 sam le 0.09 12.0 th us 1.50 3' dA 17,846 6,562 sam le 0. 33.3 th us 1.50 blunt; 28,722 17,438 reference_ _ _ -__ 0.75 blunt: 28,328 17,044 reference- -th us th us 0.38 blunt: 21,980 10,698 reference- -th us 0.19 blunt _ 4,325 reference- -15,609 no DNA - - 11,284 0 blank - -Example 3 FLM-PCR Amplification Using One FRET -Labeled Oliaonucleotide and Two Unlabelled Olit~onucleotides Introduction:
The FLM-PCR method of Example 3 is schematically illustrated in Figure 1.
Materials and Methods:
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 the same reaction sequence. The first set of oligonucleotides was SEQ ID N0:7, SEQ ID NO:10, and SEQ ID NO:1I. PCR controls contained either no DNA or no Taq polymerase.
Additionally, four oligonucleotides consisting of the following sequences were synthesized: 5'-TGCGGTGAGAGTG-(3'-C7-amine) (SEQ ID N0:14), 5'-ACTGAACCTGACCGTACACTCTCACCGCA-3' (SEQ ID NO:15), 5'-(fluorescein)-ACCGATGCGTTCGAGCATCGG-(DABCYL)-TACTGAACCTGAC'CGTACA-3' (SEQ ID N0:16), and 5'-(fluorescein)-AGCTGGAACTGTA.TCCAGCT-(DABCYL)-ACTGAACCTGACCGTACA-3' (SEQ ID
NO:1).

The second set of oligonucleotides consisted of SEQ ID N0:14, SEQ ID N0:15, and SEQ ID N0:16. Identical sequence segments of SEQ ID N0:15 and SEQ ID N0:16 are underlined, and the annealing relationships of SEQ ID N0:14 and SEQ ID
N0:15 are illustrated in Figure 1, in which a 3' mark denotes a modified 3' end. The third set of oiigonucleotides consisted of SEQ ID N0:14, SEQ ID N0:15, and SEQ ID NO:1.
Identical sequence segments of SEQ ID N0:15 and SEQ ID NO:1 are underlined SEQ ID NO:lEi and SEQ ID NO:1 each had a single-stranded stem that was less prone to primer-dimer formation than a single-stranded stem in SEQ ID NO:11.
SEQ ID
N0:16 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:1 gave the highest ratio of sample/control fluorescence.
Table 5 fluorescence 0s/sec) counts ( 10 ~n Labeled pCR CyclesControl Sample S~ple/

Primer Control 1 SEQ ID 35 11 37 3.4 NO:16 1 SEQ ID 24 26 70 2.7 NO:11 2 SEQ ID 24 13 50 3.8 NO:11 NO:1 3 SEQ ID 24 7 41 5.9 NO:1 4 SEQ I~ 30 6 35 5.8 NO:1 Example 4 Calibration of FLM-PCR for Quantitation of Blunt DNA Ends Introduction:
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 that of the genomic DNA. This condition generally can be satisfied if the standard and the genomic DNA are both ligated with any complementary ended duplex having the 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.
Optionally, the sequence of the linker-primer oligonucleotide that is ligated to any kind of standard can be made different from the sequence of the linker-primer oligonucleodde that is ligated to genomic DNA. This difference confers the potential to use differently FRET-labeled oligonucleotides with different primer sequences in the 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 both kinds of amplicons at the same time.
Standards for two different uses are described. The invention relates to a comparison of data obtained from either a parallel or a multiplex reaction comprising one or more standards. T'he standard calibrator and its ancillary oligonucleotides preferably may be included in a kit of the 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. Such a standard is likely to contain a very wide range of DNA fragment sizes, each of which will be amplified with 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 the range of about 150-2000 bp, and having only one specific kind of end. Such 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 pg DNA when reacted to completion, if the plasmid and the 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 method, it may be difficult to provide a single standard calibrator comprising all equivalent ends. Also, the highest quality can be obtained using commercially available restriction enzymes if those forming blunt-ended fragments are selected. By varying the amount of either the calibrator DNA
strand or the genomnic DNA strand, these can be titrated to a range near an equivalence point at which the respective fluorescent signals would represent nearly equal numbers of DNA ends, and then the analyte is quandtated by comparison to the calibrator curve.
Materials and Methods:
Calibrator 1 comprised blunt-ended DNA fragments made by cutting the plasmid SV40 with the endonu.clease 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-enckd DNA fragments made by cutting the plasmid LITMUS 28 with the endonuclease Mspl I, from which four fragments were expected (292 bp, 1724 bp, 1969 by and 2708 bp). Enzyme, plasmid, and buffer were purchased from New England Biolabs. Calibrator 3 comprised fragments with a on~base 3' overhanging end made by cutting the 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 P,1 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 °b ) 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 fmzen until use. They were diluted into PCR
reactions with SEQ II) NO:1, 0.5 ~M.
Results:
All of the systems tested gave a range of fragments of the expected sizes, though some fragments of nearly the same size could not be resolved on the gels used.
A standard religation and electrophoresis assay was done on the fragments of calibrators 2 and 3. The blunt ended fragments appeared to have been largely religated to form many new bands on the gel, while the overhanging ended fragments did not.
Ligated blunt-ended fragments were amplified by PCR and gave a fluorescent signal well above controls. The SV40 standard gave a linear calibration curve over the range of 0.25 to 2 ng DNA/20 ~.1 reaction volume. R2 was 0.96 using data at 0 (triplicate), 0.25, 0.5, 1.0, and 2.0 ng (duplicates). The signal/blank ratio was 5.8 at 2 ng DNA
and 1.9 at 0.25 ng DNA (detector background subtracted). This calibrator is useful for quantitating sample DNA ends, for if all standard fragments were amplified to the same extent, 3.4 pmol of DNA ends per microgram plasmid would have been expected. A more accurate calculation would take into account the actual ratios of fragment sizes in the amplicon.
Example 5 An Internal Standard Using a Second Color for Multiplex PCR
Introduction:
The present invention includes a "multiplex PCR" method 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 oligonucleodde is labeled with a second MET pair, e.g., Texas Red (or rhodamine) and DABCYL that is different from the first MET pair. Multiplex PCR is used for the detection of two distinct amplification reactions in a single reaction vessel.
Any two multiplexed reactions should be performed using oligonucleotides of nearly identical secondary structures and physicochemical characteristics, differing only in their MET labels and oligonucleodde sequences. Multiplexation confers the advantage of identical reaction conditions and measurement conditions for the two reactions, significantly increasing the accuracy of measurement. Because PCR is a method for geometric amplificatian, measurable differences can arise between samples due to even small differences in their internal conditions during thermal cycling.
A very accurate method of calibration involves ligation of a portion of a standard calibrator with an oligonucleotide differing sufficiently in sequence from the oligonucleotide that 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. Thus, 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 than the sequence to which the 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 the relative amount of DNA per sample is consistent and reproducible. A simple method for estimation of the relevant statistical measures is to amplify a specif c 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 another pair of MET labeled primers that have been optimized to amplify under the same reaction conditions as the genomic DNA.
Two examples of genes that are known in the art to be amenable for this use are the glyceraldehyde-3-phosphate dehydrogenase gene and the actin gene.
Materials and Methods:
One set of oligonucleotides is SEQ ID N0:14, SEQ ID NO:15, and SEQ ID NO:1.
A second set of oligonucleotides is chosen to have the same length, the same annealing temperature, and similar percentages of purine and pyrimidine bases as the first set. The analog of SEQ ID NCI:1 is labeled with a different fluorophore and quencher, such as rhodamine and DABC;YL. This similarity allows one to do PCR amplification of both 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 the 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 the measurements are within a statistically validated zone of the assay, the proportionality of the sample to the standard allows for doing a measurement of the amount of sample ends.
This application claims priority from provisional U.S. Application Serial No.
60/069,434 filed December 12, 1998, the entire contents of which are herein incorporated by reference.

Claims

WE CLAIM:

1. A method for detecting a double-stranded nucleic acid fragment comprising:
(a) providing a linker-primer duplex containing first and second oligonucleotides, wherein the first oligonucleotide is annealed to the second oligonucleotide;
and providing a third 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, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein 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 hairpin; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed, (b) ligating the 3' end of the first oligonucleotide to the 5' end of the first strand of the fragment to form a ligated first strand, and ligating the 3' end of the first oligonucleotide to the 5' end of the second strand of the fragment to form a ligated second strand, (c) extending the 3' end of the ligated first strand using the ligated second strand as a template to form an extended first strand, and extending the 3' end of the ligated second strand using the ligated first strand as a template to form an extended second strand, wherein the extended first strand is annealed to the extended second strand, (d) separating the extended first strand from the extended second strand, (e) annealing the third oligonucleotide to each of the extended first and second strands, (f) extending the 3' end of the third oligonucleotide using the extended first strand as a template to form a labeled second strand, and extending the 3' end of the extended first strand using the third oligonucleotide as a template to form a doubly extended first strand, wherein the labeled second strand is annealed to the doubly extended first strand; and extending the 3' end of the third oligonucleotide using the extended second strand as a template to form a labeled first strand, and extending the 3' end of the extended second strand using the third oligonucleotide as a template to form a doubly extended second strand, wherein the labeled first strand is annealed to the doubly extended second strand, (g) separating the labeled first strand from the doubly extended second strand, and separating the labeled second strand from the doubly extended first strand, (h) annealing the third oligonucleotide to each of the labeled first and second strands, and to each of the doubly extended first and second strands, (i) extending the 3' end of the third oligonucleotide using the labeled first strand and the doubly extended first strand as templates to form extended labeled second strands; and extending the 3' end of the third oligonucleotide using the labeled second strand and the doubly extended second strand as templates to form extended labeled first strands, (j) optionally amplifying the extended labeled first and second strands, and (h) detecting the emitted energy to detect the fragment.

2. The method of claim 1, wherein (j) comprises:
(i) separating the extended labeled first strand from the extended labeled second strand, (ii) annealing the third oligonucleotide to each of the extended labeled first and second strands, (iii) extending the 3' end of the third oligonucleotide 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;
and extending the 3' end of the third oligonucleotide 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, and (iv) repeating (i), (ii), and (iii) 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).

3. The method of claim 1, wherein the donor moiety is a fluorophore and the acceptor moiety is a quencher of light emitted by the fluorophore.

4. The method of claim 1, wherein the donor moiety is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate, and Reactive Red 4; and the acceptor moiety is selected from the group consisting of DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, fluorescein, Malachite green, and Texas Red.

5. The method of claim 1, wherein the donor moiety is fluorescein, and the acceptor moiety is DABCYL.

6. The method of claim 1, wherein the donor and acceptor moieties are respectively attached to complementary first and second nucleotides in the hairpin.

7. The method of claim 1, wherein the 3' end of the second oligonucleotide cannot be extended by a nucleic acid polymerase.

8. The method of claim 1, wherein a phosphate group is not attached to the 3' end of the first oligonucleotide, and a phosphate group is not attached to the 5' end of the second oligonucleotide.

9. The method of claim 1, wherein the third oligonucleotide consists of the sequence 5'-AGCTGGAACTCTATCCAGCTACTGAACCTGACCGTACA-3' (SEQ ID NO:1), wherein fluorescein is attached to the residue at position 1 and DABCYL is attached to the residue at position 20.

10. The method of claim 1, wherein 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 5'-GTGTACGGTCAGG-3' (SEQ ID
NO:6).

11. The method of claim 1, wherein an end of the fragment is a blunt end, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a blunt end in the duplex.

12. The method of claim 1, wherein an end of the fragment has a terminal overhang, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.

13. The method of claim 1, wherein the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands.

14. The method of claim 1, wherein the emitted energy detected in (j) is light.

15. The method of claim 1, wherein the fragment is produced in a cell as a result of apoptosis.

16. A method for detecting a double-stranded nucleic acid fragment comprising:
(a) providing a linker-primer duplex containing first and second oligonucleotides, 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 comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein 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 hairpin; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed, (b) ligating the 3' end of the first oligonucleotide to the 5' end of the first strand of the fragment to form a labeled first strand, and ligating the 3' end of the first oligonucleotide to the 5' end of the second strand of the fragment to form a labeled second strand, (c) extending the 3' end of the labeled first strand using the labeled second strand as a template to form an extended labeled first strand, and extending the 3' end of the labeled second strand using the labeled first strand as a template to form an extended labeled second strand, wherein the extended labeled first strand is annealed to the extended labeled second strand, (d) optionally amplifying the extended labeled first and second strands, and (e) detecting the emitted energy to detect the fragment.

17. The method of claim 16, wherein (d) comprises:
(i) separating the extended labeled first strand from the extended labeled second strand, (ii) annealing the first oligonucleotide to each of the extended labeled first and second strands, (iii) extending the 3' end of the first oligonucleotide 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;
and extending the 3' end of the first oligonucleotide 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, and (iv) repeating (i), (ii), and (iii) 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).

18. The method of claim 16, wherein an end of the fragment is a blunt end, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a blunt end in the duplex.

19. The method of claim 16, wherein an end of the fragment has a terminal overhang, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.

20. The method of claim 16, wherein the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands.

21. The method of claim 16, wherein a phosphate group is not attached to the 3' end of the first oligonucleotide, and a phosphate group is not attached to the 5' end of the second oligonucleotide.

22. The method of claim 16, wherein the fragment is produced in a cell as a result of apoptosis.

23. A method for detecting a double-stranded nucleic acid fragment comprising:
(a) providing a linker-primer 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 comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein the first nucleotide sequence is annealed to the third nucleotide sequence to form a hairpin; 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; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed, (b) ligating the 3' end of the. oligonucleotide to the 5' end of the first strand of the fragment to form a labeled first strand, and ligating the 3' end of the oligonucleotide to the 5' end of the second strand of the fragment to form a labeled second strand, (c) extending the 3' end of the labeled first strand using the labeled second strand as a template to form an extended labeled first strand, and extending the 3' end of the labeled second strand using the labeled first strand as a template to form an extended labeled second strand, wherein the extended labeled first strand is annealed to the extended labeled second strand, (d) optionally amplifying the extended labeled first and second strands, and (e) detecting the emitted energy to detect the fragment.

24. The method of claim 23, wherein (d) comprises:
(i) separating the extended labeled first strand from the extended labeled second strand, (ii) annealing the oligonucleotide to each of the extended labeled first and second strands, (iii) extending the 3' end of the oligonucleotide 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 extend labeled second strand; and extending the 3' end of the oligonucleotide 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, and (iv) repeating (i), (ii), and (iii) 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).

25. The method of claim 23, wherein an end of the fragment is a blunt end, and the 3' end of the first sequence and the 5' end of the third sequence form a blunt end in the oligonucleotide.

26. The method of claim 23, wherein an end of the fragment has a terminal overhang, and the 3' end of the first sequence and the 5' end of the third sequence form a terminal overhang in the oligonucleotide that is complementary to the terminal overhang in the fragment.

27. The method of claim 23, wherein the 5' end of the oligonucleotide cannot be ligated to the 3' end of each of the first or second strands.

28. The method of claim 23, wherein a phosphate group is not attached to each of the 5' and 3' ends of the oligonucleotide.

29. The method of claim 23, wherein the fragment is produced in a cell as a result of apoptosis.

30. A method for detecting a double-stranded nucleic acid fragment comprising:
(a) providing a linker-primer duplex containing:
(i) a first oligonucleotide, (ii) a second oligonucleotide, and (iii) a molecular energy transfer pair comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein the first oligonucleotide is annealed to the second oligonucleotide;
the first oligonucleotide contains the donor moiety and the second oligonucleotide contains the acceptor moiety; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the linker-primer duplex is formed, (b) ligating the 3' end of the first oligonucleotide to the 5' end of the first strand of the fragment to form a labeled first strand, and ligating the 3' end of the first oligonucleotide to the 5' end of the second strand to form a labeled second strand, (c) extending the 3' end of the labeled first strand using the labeled second strand as a template to form an extended labeled first strand, and extending the 3' end of the labeled second strand using the labeled first strand as a template to form an extended labeled second strand, wherein the extended labeled first strand is annealed to the extended labeled second strand, (d) optionally amplifying the labeled extended ligated first and second strands, and (e) detecting the emitted energy to detect the fragment.

31. The method of claim 30, wherein (d) comprises:
(i) separating the extended labeled first strand from the extended labeled second strand, (ii) annealing the first oligonucleotide to each of the extended labeled first and second strands, (iii) extending the 3' end of the first oligonucleotide 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;
and extending the 3' end of the first oligonucleotide 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, and (iv) repeating (i), (ii), and (iii) 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).

32. The method of claim 30, wherein an end of the fragment is a blunt end, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a blunt end in the duplex.

33. The method of claim 30, wherein an end of the fragment has a terminal overhang, and the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide form a terminal overhang in the duplex that is complementary to the terminal overhang in the fragment.

34. The method of claim 30, wherein the 5' end of the second oligonucleotide cannot be ligated to the 3' end of each of the first and second strands.

35. The method of claim 30, wherein a phosphate group is not attached to the 3' end of the first oligonucleotide, and a phosphate group is not attached to the 5' end of the second oligonucleotide.

36. The method of claim 30, wherein the fragment is produced in a cell as a result of apoptosis.

37. A kit comprising:
(a) ligase, (b) a first oligonucleotide, (c) a second oligonucleotide capable of annealing to the first oligonucleotide; and (d) a third 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, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein 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 hairpin; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.

38. A kit comprising:
(a) ligase, (b) a first 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, (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence, and (v) a molecular energy transfer pair comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, and (c) a second oligonucleotide, wherein 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 hairpin; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.

39. A kit comprising:
(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 comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein the first nucleotide sequence is capable of annealing to the third nucleotide sequence to form a hairpin; 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; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the hairpin is formed.

40. A kit comprising:
(a) ligase, (b) a first oligonucleotide, (c) a second oligonucleotide, and (d) a molecular energy transfer pair comprising an energy donor moiety capable of emitting energy, and an energy acceptor moiety capable of absorbing the emitted energy, wherein the first oligonucleotide is capable of annealing to the second oligonucleotide; the first oligonucleotide contains the donor moiety and the second oligonucleotide contains the acceptor moiety; and the acceptor moiety absorbs a substantial amount of the emitted energy only if the linker-primer duplex is formed.