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WO2003046512A2 - Procede permettant l'amplification de signaux de titrage biologique moleculaires - Google Patents

Procede permettant l'amplification de signaux de titrage biologique moleculaires Download PDF

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WO2003046512A2
WO2003046512A2 PCT/US2002/038104 US0238104W WO03046512A2 WO 2003046512 A2 WO2003046512 A2 WO 2003046512A2 US 0238104 W US0238104 W US 0238104W WO 03046512 A2 WO03046512 A2 WO 03046512A2
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Prior art keywords
amplifier
amplification
probe
primers
target
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WO2003046512A3 (fr
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Fengchun Ye
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Royce Technologies LLC USA
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Royce Technologies LLC USA
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Priority claimed from CA002363703A external-priority patent/CA2363703A1/fr
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Publication of WO2003046512A2 publication Critical patent/WO2003046512A2/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a method for covalent linkage of a specially designed sequence to probing molecules used in bio-assays/detection reactions, and subsequent amplification of this sequence to fortify the initial signals from the target-bound probing molecules in the bio-assays.
  • the disclosed method is a very useful technique that can have wide applications in molecular biology, chemistry, biotechnology, medical diagnosis, and forensic science.
  • a big challenge in molecular detection or other molecular interaction based bio-assays is low sensitivity of the detection system. Very often, it is necessary to amplify the detecting signals in order to detect target molecules of extremely low quantity.
  • a means of amplifying nucleic acid molecules is particularly of great value because a lot of molecular detection methods and bio-assays use molecular hybridization of nucleic acids (DNA-DNA, DNA-RNA, or RNA-RNA), including Dot blot hybridization, Southern and Northern blot hybridization, in situ hybridization, and DNA chip screening, etc.
  • Applications of nucleic acid detection are extremely broad, ranging from fundamental gene mapping, genotyping, and gene expression analysis, to diagnosis of infectious diseases, cancer, and genetic diseases.
  • nucleic acid molecules can be easily conjugated to other molecules such as proteins. Conjugation of nucleic acid to other molecules is essential for their own detection, and/or for having complementary and fortified signals when such molecules themselves are used as probes in detection.
  • PCR polymerase chain reaction
  • dNTP dATP, dCTP, dGTP, and dTTP
  • the reaction begins with converting double-stranded target DNA into single-stranded DNA under denaturing conditions (high temperature).
  • LCR ligase chain reaction
  • DNA amplification methods that are performed under isothermal (37 °C) conditions have also been developed. These include self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Q.beta replicase, and rolling circle amplification (RCA) methods (Fahy, et al., PCR Methods, 1:25-33 (1991). Guatelli, et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878. Compton, Nature, 359: 91-92 (1991). Walker, et al., Nucleic Acids Res., 20: 1691-1696 (1992).
  • 3SR self-sustained sequence replication
  • NASBA nucleic acid sequence based amplification
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • the enzymes are used to amplify specific RNA and/or DNA target molecules exponentially.
  • the SDA method is based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand.
  • the displaced sense strand serves as a target for an antisense reaction and vice versa, resulting in exponential growth of the target molecule.
  • the RCA method mimics the DNA replication mechanism of some viruses.
  • a DNA polymerase, primed by a primer reads off of a single promoter around a circle of DNA. This continuously rolls out linear stretches of the circle. At first it creates a copy of itself, then it continues to create a concatenated string of multiple copies.
  • LRCA linear RCA reaction
  • ERCA exponential RCA
  • HRCA strand displacement reactions
  • RCA has some advantages over these methods, such as covalent linkage of all amplified signals to the target, and the ability to amplify very large molecules. Nevertheless, all these in vitro amplification procedures require functional enzymes to replicate the target molecules. These enzymes can be difficult and costly to produce, and the amplification reactions are fairly complicated. In addition, except RCA, all other methods are very difficult to be used for detection /bio-assay involving other types of molecules such as proteins.
  • reagents and a method for efficient in vitro amplification of signals in molecular detections such as nucleic acid hybridization, antibody/antigen immunoassay, or other specific molecule-molecule binding assays.
  • the method uses three oligonucleotide primers.
  • the first primer referred to as reference primer, is 25-50 bases long in general, and is covalently linked to the probe molecules (DNA, RNA, antibody, etc.) used in the detection/bio-assays.
  • the second primer, Amplifier I. is a symmetrical molecule, with its 5' half sequence fully complementary to the reference primer and its 3' half sequence fully complementary to the 3' half of a third primer named Amplifier II.
  • Amplifier LI is also symmetrical, with its 5' half identical to the reference sequence and thus also fully complementary to the 5' half of Amplifier I.
  • the two amplification primers are also compatibly labeled. This labeling can be done using either radioactive or non-radioactive methods such as fluorescence, biotin, and others.
  • the signal amplification procedure makes use of the ability of the two "end-to-end” complementary amplification primers to hybridize and form highly repetitive sequences. It follows the last wash step of the molecular detection assays.
  • Amplifier I is first incubated and hybridized with the reference primer linked to the probe molecules that are specifically bound to the targets.
  • Amplifier II is then added, which hybridizes with the 3' half of amplifier I.
  • the protruding single-stranded 5' half of the added Amplifier II will continue to hybridize with the 5' half of Amplifier I in the buffer, and the "walk” continues endlessly until the incubation is stopped.
  • the amplification is not exponential, the "linear addition” process can effectively amplify the detection signals hundreds of times within a short period.
  • Non-Enzymatic Amplification is a simple but efficient procedure for signal amplification. It does not require any enzyme to amplify the involved nucleic acid molecules, and can be used for signal amplification of any bio-assay that is based on molecular interactions. These molecular interactions include those between DNA-DNA, DNA-RNA, RNA-RNA, DNA/RNA- protein, protein-protein, molecule-cells, and any other probe-target interactions. Thus, the method can be used to amplify signals of a variety of different probe molecules, including nucleic acids, peptides, proteins, and other chemicals.
  • the method can be used to simultaneously amplify signals of multiple probes/ targets in a single detection/bio-assay.
  • Another important distinction between all the previously described amplification methods and the method of the present invention is that the latter does not amplify the target molecule itself. Rather, it amplifies additive nucleic acid signals that are integrated and added to the target-specific probing molecules.
  • the present invention has the advantages of being highly useful in much broader applications, involves an easier procedure, and has lower cost, no risk of contamination, and more flexibility, especially in-molecular detection and diagnosis assays.
  • FIG. 1 depicts the designs of the reference primer, the Amplifier I primer, and the
  • FIG. 2 illustrates steps of the amplification procedure.
  • FIG. 3 demonstrates how the method amplifies signals in detections using nucleic acid hybridization.
  • FIG. 4 demonstrates how the method amplifies signals in detections using antibody or other proteins as probes.
  • a "target” in a molecular detection/bio-assay refers to the molecule or the organism in a testing sample that the assay tries to identify and/or quantify. It can be a molecule, such as nucleic acid, protein, peptide, carbohydrate, lipid, hormone, antibody, antigen, and a chemical; it can also be an organism, such as a virus, bacterium, or cell, etc.
  • a “probe” in a bio-assay refers to the molecule that the assay uses to specifically identify and/or qualify the target in a testing sample.
  • the probe is usually a molecule, such as nucleic acid, protein, peptide, carbohydrate, lipid, hormone, derivatives thereof or analogs thereof, that has intrinsic capabilities to specifically bind to its target.
  • the probe molecules are usually labeled with a fluorescent label, a phosphorescent label, an enzymatic label, a chemical label (such as biotin or digoxigenin), or a radioactive label, for specific signal detection.
  • a “oligonucleotide” primer refers to a sequence-defined and length-defined nucleic acid or analog thereof.
  • the disclosed method is a simple but very effective procedure to substantially amplify signals in molecular detection. Unlike other DNA amplification technologies, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and rolling cycle amplification (RCA), etc., the disclosed method does not use any enzyme such as DNA polymerase or DNA ligase.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RCA rolling cycle amplification
  • the disclosed method requires three nucleic acid primers: a reference primer and two amplification primers (Amplifier I and Amplifier IT). None of these primers should have significant homo logy with the probe/target molecules in nucleic acid hybridization assays. However, in other methods of molecular detection such as immunoassays, this requirement does not apply, and a universal set of primers can be used.
  • the first primer generally 25-50 bases long (note: longer sequences might also be used, as far as they can be produced).
  • This can be either a DNA or RNA sequence.
  • This sequence should be relatively G+C rich, without any ability to form internal "hair-pin" structures.
  • This sequence might be derived from the "multiple cloning sites" of a DNA cloning vector, in which an interested DNA fragment is inserted.
  • the reference primers can be chosen from the two regions near the two ends of the cloned DNA fragment insert. When the interested DNA fragment is cut out and used as a probe for molecular hybridization, the selected reference primers from the vector sequences should be included and attached to the probe DNA fragment.
  • cRNA probe preparation when using the insert-carrying plasmid as a template for in vitro transcription with T3, T7 or SP6 RNA polymerase.
  • a reference sequence can be included and attached to each probe molecule during primer synthesis.
  • the reference primer sequence can be attached to the 5' end of the polyT primer that is used in the reverse transcription reaction. As such, the resulting cDNA molecules (probe) will have the reference primer sequence attached at their 5' end.
  • the selected reference primer should be covalently immobilized to the probe molecules.
  • a number of strategies can be used for covalent coupling of nucleic acid molecules to proteins. Ultraviolet cross-linking can effectively immobilize DNA molecules to proteins (Jang, et al., J. Immuno. 145: 3353-3359 (1990).). Chemical cross-linking also can be very efficient. One of these methods is coupling 5' Thiol oligos to sulfo-GMBS activated and ⁇ -mercaptoethanol treated proteins (Schweitzer, et al., Proc. Natl. Acad. Sci.
  • Another method is coupling 5' amine-modified oligos to proteins through an amine crosslinker reagent.
  • Glutaraldehyde can also be used to couple 5 'amine-modified oligos to proteins.
  • the oligonucleotide conjugated proteins are finally subjected to gel filtration purification to remove free oligonucleotides.
  • Coupling DNA primers to other types of probe molecules is also possible. Chemical reactions depend on the nature of the probe molecules.
  • the other two primers in the disclosed method are amplification primers: Amplifier I and Amplifier II. Depending on the reference primer, these primers are generally 50-100 bases long. They can be either DNA or RNA sequences. Again, longer sequences can be used for the amplification process, as long as they can be produced. These sequences can be either synthetic primers, can be made through recombinant DNA technology, or can be the products of in vitro transcription or reverse transcription reactions.
  • the primer Amplifier I is a symmetrical molecule, with its 5' half sequence fulLy complementary to the reference primer, and its 3' half sequence fully complementary to the 3' half of the third primer, Amplifier II. W hen choosing the primer sequences, one must make sure that there isn't any possibility for the primers to form internal "hair-pin" structures. Like Amplifier I, Amplifier II is also symmetrical, and its 5' half is identical to the reference sequence and fully complementary to the 5' half of Amplifier I.
  • the two amplification primers are compatibly labeled. For instance, if the probe is given a radioactive label such as P, the two amplification primers should also be labeled with the same radioactive element. This will ensure that the amplified signals and the original signals from the probe molecules are entirely compatible and can be detected by the same method or device. This makes it possible for the two readings to be added together.
  • labeling systems can be either radioactive methods or non-radioactive methods that use fluorescence, biotin, and other labeling chemicals.
  • T4 polynucleotide kinase or terminal deoxynucleotidyl transferease can be used for end labeling.
  • fluorescence, biotin, digoxigenin, or other labeling molecule- conjugated nucleotides can be directly incorporated into the sequence during the primer synthesis.
  • the signal amplification procedure follows the last wash step of the molecular detection assays. At this point, only the probe molecules are specifically bound to the targets.
  • Amplifier I is first incubated with the "probe-target" complex, hybridizing with the reference primer that has been linked to the probe molecules in the previous steps. This step can be done in a regular DNA hybridization buffer or in a particular buffer compatible with the bio-assays. The key is that all solutions must be nuclease-free. This step can take place within a few minutes. Amplifier II is then added, which hybridizes with the 3' half of Amplifier I.
  • the protruding single-stranded 5' half of the added Amplifier II will continue to hybridize with the 5' half of free Amplifier I in the hybridization solution.
  • the outcome is the formation of a double-stranded hybrid molecule with another protruding single-stranded 3' half of Amplifier I, which will continue to hybridize with free Amplifier II.
  • the "walk" continues endlessly until the incubation is stopped.
  • the amplification process is done in a programmed manner, with the two amplification primers separately added to the amplifying molecule.
  • the two amplification primers are "end-to-end” complementary sequences.
  • Co-incubation of these two primers may generate linear and circular hybrids of various sizes in the hybridization solution. Consequently, this may not be in favor of the continuous addition of the two primers to the extending hybrid (amplifying signal) attached to the "probe- target" complex.
  • separate incubation of the "probe-target” complex with the two amplification primers would keep the two primers constantly at high concentration. This would facilitate the hybridization and extension of the hybrid bound to the "probe-target” complex.
  • a disadvantage of such separate addition is that longer time is required to have a substantial amplification because each addition step takes at least one minute.
  • the whole amplification process may be done with the two primers mixed in a single tube and carried out as a single step. In this case, incubation time can be largely reduced.
  • the amplification is not an exponential but a linear addition process, it can effectively amplify the detection signals hundreds of times within a short period.
  • Tm of all the primers must be calculated for different salt concentrations.
  • Stringency conditions mainly temperature and salt concentration
  • All steps in signal amplification are carried out under sufficiently stringent conditions.
  • the disclosed method is basically a non-enzymatic process, we have found that the reaction, when conducted in an appropriate buffer, can be significantly improved in the presence of a DNA ligase, such as T4 DNA ligase. Therefore, whenever possible, it is suggested to include a certain amount of DNA ligase in the solutions containing the two amplification primers, in order to achieve higher signal amplification.
  • This kind of application includes a number of molecular hybridization bio-assays, such as Southern blot, Northern blot, in situ hybridization on tissue, cells, or chromosomal spreads, and molecular hybridization on DNA chips, etc.
  • the disclosed method can be particularly useful when the detection signals are very weak without signal amplification.
  • Fig. 1 A illustrates how DNA, RNA, and oligonucleotide probes can be prepared to have the reference primer(s) attached to one or both ends.
  • the probe is made from a DNA fragment cloned in a plasmid or phage vector, it would be advantageous to have two reference primers, each attached to one end of the insert (probe DNA). This would allow simultaneous signal amplification at both ends of the probe DNA molecule.
  • the two reference primers can be chosen from the vector sequences near the two ends of the probe DNA, in the multiple cloning sites (MS) regions.
  • a radioactively or non- radioactively labeled DNA probe can be prepared in the presence of a labeled-dNTP (dCPT, dGTP, dATP, or dTTP) by PCR amplification of the chosen area. Otherwise, the chosen fragment (including the reference sequences at both ends of the insert) can also be cut out of the vector with appropriate restriction enzymes, and labeled with whatever appropriate methods.
  • cR A (complementary RNA) probes can also be made if promoter sequences for T3, T7, or SP6 RNA polymerases are available upstream of the multiple cloning sites from either direction.
  • mRNAs messenger RNAs with polyA tails
  • a randomly formulated reference primer can be directly attached to a polyT sequence. It can be attached at its 5 'end during oligo primer synthesis. This polyT primer is then used to prime first-strand cDNA synthesis, in the presence of a labeled-dNTP and reverse transcriptase. Finally, if synthesized oligo primers are used as probes, the reference primer can be directly attached to the probe primer during primer synthesis and labeling.
  • Fig. IB illustrates steps of molecular hybridization and signal amplification after hybridization.
  • a further signal amplification step is carried out using the disclosed method. Since the two reference primers are different sequences, one way to do signal amplification at both ends of the probe molecule is to have two pairs of amplification primers, with one pair fitting one reference primer and the other pair fitting the other reference primer.
  • the advantage of this strategy is that there will not be any interference between the amplifying signals at the two ends.
  • the disadvantage is that it requires four amplification primers. To avoid such complication, another strategy involves the use of a single pair of amplification primers that are designed as follows:
  • Amplifier I its 5' half to be complementary to the reference primer at the 5' end of the probe molecule, and its 3' half to be complementary to the reference primer at the 3' end of the probe molecule;
  • Amplifier II its 5' half to be identical to the reference primer at the 5' end of the probe, and its 3' half to be identical to the reference primer at the 3' end of the probe.
  • the advantage of this strategy is that it requires only one pair of amplification primers.
  • the disadvantage is that at some point the extended hybrids from the two ends of the probe may form a closed circle, preventing further extension of the amplifying signals. This occurs because the two amplification primers are "end-to-end” complementary. From this point of view, the "four-primer" strategy is a better choice.
  • the two different Amplifier I primers can be mixed and prepared as one solution, and the two different Amplifier II primers can be mixed and prepared as the second solution. It is best not to mix the four primers together during the amplification process. This is because, as described in the "Designs of Primers" section, mixing Amplifier I and Amplifier II would allow them to form hybrids in the solution, which reduces the concentration of the primers going to the "target-probe" complex for signal amplification. With the two solutions, signal amplification is carried out by alternately incubating the "target-probe” complex with each solution for approximately one to two minutes, and repeating the "two-step” amplification for as many cycles as possible. This can be done in an automatic manner.
  • the target After stopping amplification and washing away non-specific binding under stringent conditions, the target is revealed.
  • This can be realized through exposure to X-ray film when radioactive labeling or chemiluminescence detection is used. Alternatively, it can be realized by enzyme-substrate reaction when non-radioactive labeling is used or by fluorescence microscopy reading when fluorescent labeling is used.
  • bio-assays and molecular detection techniques are based on immunochemical reactions, particularly antibody-antigen interactions. These include protein analysis by Western blotting, antigen detection by ELIS A and/or immunohistochemical staining, and the recently developed protein chip technology, etc.
  • a big challenge for protein-based bio-assays is how to increase their sensitivity to be able to detect the lowest amount of the target molecules.
  • Fig. 4 illustrates how the disclosed method can be used to amplify signals in an immunochemical assay (antigen detection using a specific antibody).
  • a reference primer must be covalently linked to the probe, the antigen-specific antibody molecule. This can be realized a number of ways, including chemical cross-linking using either 5' amine-modified oligo primer or 5' thiol-modified oligo primer (see description in the "Designs of Primers" section).
  • an immunoassay (Fig. 4B or C) can be conducted by incubating the antibody with the target (antigen). Prior to the incubation, this latter might have been immobilized onto a solid-support such as a membrane (Western blot), or captured by a specific antibody that had been immobilized on the surface of a plastic plate (ELIS A) or slide (protein chips). After incubating and washing away non-specific binding, the "antigen-antibody” complex is detected by a secondary antibody that is raised against the first (antigen-specific) antibody in a different animal species. This secondary antibody is either conjugated to a functional enzyme, such as alkaline phosphatase or horseradish peroxidase, or to a fluorescent molecule or other labeled molecule.
  • a functional enzyme such as alkaline phosphatase or horseradish peroxidase, or to a fluorescent molecule or other labeled molecule.
  • the secondary antibody is directly labeled with a radioactive or fluorescent label (Fig. 4C)
  • the Amplifier I and Amplifier II primers must be labeled the same way.
  • the target can be detected on X-ray film or under fluorescence microscope/scanner.
  • Other application examples of the disclosed method include ligand-receptor binding assays, adhesin-cell binding assays, etc. Regardless of the nature of the assays, it is important to note that it is always the probe molecules that need to be conjugated to the reference primer for signal amplification. Methods to conjugate nucleic acid molecules to the probe molecules depend on the chemical nature of the probes. The same amplification procedure used for nucleic acid hybridization (C) and immunochemical detection (D) can be followed.
  • bio-assays particularly in situ detection and immunohistochemical staining, use multiple probes that are differentially labeled to simultaneously detect different targets.
  • a separate set of reference primer and amplification primers is specifically designed for each of the probes. Also it is important that each set of amplification systems and its corresponding probe for signal amplification must be fully compatibly labeled.
  • target 1 is cytoplasmic mRNA of gene X
  • target 2 is cytoplasmic mRNA of gene Y
  • a cDNA probe for gene X mRNA is labeled with fluorescent Cy3 and attached at one or both ends with a reference primer for signal amplification.
  • the amplification primers, Amplifier I and Amplifier II, for this probe must also be labeled with Cy3.
  • a cDNA probe for gene Y mRNA is labeled with fluorescent Cy5 and attached at one or both ends with a different reference primer.
  • the amplification primers for gene Y probe are also labeled with Cy5. Signal amplification for both probes can be done simultaneously.
  • the two differentially labeled Amplifier I primers are mixed as one hybridization buffer, and the two differentially labeled Amplifier II primers are mixed as another hybridization buffer.
  • the sample target-probe complexes
  • the sample is then subjected to the two buffers alternately for multiple cycles of amplification, under sufficiently stringent hybridization conditions. After washing, the Cy3 and Cy5 signals can be scanned and detected with different wavelengths.
  • the same strategy can be used for overall differential gene expression monitoring using DNA chips.
  • Such a test is widely used to monitor overall changes in gene expression of an organism or cells under a different environmental or physiological condition (experimental condition).
  • environmental condition environmental condition
  • DNA spots arrayed in a very well defined pattern, with each spot representing an individual gene.
  • total mRNAs from the sample treated under an experimental condition are converted into single-stranded cDNAs by reverse transcriptase, and are labeled with a fluorescent, such as Cy3, during the cDNA synthesis.
  • a fluorescent such as Cy3
  • an equal amount of total mRNAs from a normal sample (control) are also converted into single-stranded cDNAs, which are labeled with another fluorescent, such as Cy5.
  • the two probes are then co-incubated with the DNA chips. Genes that undergo no changes in expression are equally labeled with the two different fluorescents, whereas genes that undergo changes in expression are differentially labeled. As with other bio-assays, very often DNA chips can not detect messenger RNAs of extremely low copy numbers. In this case, signal amplification is very useful.
  • two sets of reference primer and amplification primers are needed. One reference primer is directly attached to a polyT primer for cDNA synthesis of the experimental sample, and a different reference primer is directly attached to a polyT primer for cDNA synthesis of the normal sample.
  • one set of amplification primers is labeled with Cy3 for signal amplification of the cDNA probes from the experimental sample.
  • the other set of primers is labeled with Cy5 for signal amplification of the cDNA probes from the normal sample.
  • Cy3- labeled Amplifier I (set 1) and Cy5-labeled Amplifier I (set 2) primers are mixed to form one signal amplification (hybridization) buffer
  • Cy3- labeled Amplifier II (set 1) and Cy5-labeled Amplifier II (set 2) are mixed to form the second hybridization buffer.
  • the chip After co-incubation of the two cDNA probes with the DNA chip, and after subsequent washing, the chip is subjected to the two buffers alternately for multiple cycles of signal amplification, under sufficiently stringent conditions.
  • fluorescent microscopy or a laser scanner with different wavelengths detects the two fluorescent signals.
  • probe I is an antibody that is labeled with Cy3 to detect antigen X
  • probe II is another antibody that is labeled with Cy5 to detect antigen Y.
  • each of the two antibodies is attached with a different reference primer, and each has a specific set of signal amplification primers that are compatibly labeled. Simultaneous signal amplification for two probes can be done the same way as described for differential DNA labeling.

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Abstract

L'invention a trait à des réactifs et à un procédé permettant une amplification in vitro efficace de signaux de titrage biologique. Ledit procédé fait appel à une paire d'amorces oligonucléotidiques complémentaires « bout à bout », pour former en continu une molécule hybride double brin et hautement répétitive. Etant donné qu'elle est liée par covalence et ajoutée à la molécule de sondage dans les titrages biologiques, et est également marquée de manière compatible, cette molécule hybride peut amplifier les signaux de détection des centaines de fois en un court laps de temps. Ce procédé peut avoir des applications très larges dans des titrages biologiques faisant intervenir l'hybridation moléculaire, la détection immunochimique, et d'autres interactions particulières entre deux molécules ou entre des molécules et des cellules.
PCT/US2002/038104 2001-11-23 2002-11-25 Procede permettant l'amplification de signaux de titrage biologique moleculaires Ceased WO2003046512A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002363953A AU2002363953A1 (en) 2001-11-23 2002-11-25 Method for amplification of molecular bio-assay signals

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CA002363703A CA2363703A1 (fr) 2001-11-23 2001-11-23 Methode d'amplification des signaux moleculaires pour les epreuves biologiques
CA2,363,703 2001-11-23
US10/299,066 2002-11-18
US10/299,066 US20030124604A1 (en) 2001-11-23 2002-11-18 Method for amplification of molecular bio-assay signals

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WO2009026651A1 (fr) * 2007-08-30 2009-03-05 Commonwealth Scientific And Industrial Research Organisation Méthode de détection d'un acide nucléique

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EP1098996A1 (fr) * 1998-07-20 2001-05-16 Yale University Procede pour detecter des acides nucleiques au moyen de ligature a mediation par cibles d'amorces bipartites

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026651A1 (fr) * 2007-08-30 2009-03-05 Commonwealth Scientific And Industrial Research Organisation Méthode de détection d'un acide nucléique

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