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WO1997008339A1 - Chronic myelogenous leukemia diagnostic assay - Google Patents

Chronic myelogenous leukemia diagnostic assay Download PDF

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Publication number
WO1997008339A1
WO1997008339A1 PCT/US1995/010919 US9510919W WO9708339A1 WO 1997008339 A1 WO1997008339 A1 WO 1997008339A1 US 9510919 W US9510919 W US 9510919W WO 9708339 A1 WO9708339 A1 WO 9708339A1
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WIPO (PCT)
Prior art keywords
abl2
rna
oligonucleotide
detector
solid phase
Prior art date
Application number
PCT/US1995/010919
Other languages
French (fr)
Inventor
Janice Brown
Connie Lockhart-Bruce
Original Assignee
Dade International Inc.
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Publication date
Application filed by Dade International Inc. filed Critical Dade International Inc.
Priority to AU34178/95A priority Critical patent/AU3417895A/en
Publication of WO1997008339A1 publication Critical patent/WO1997008339A1/en

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates generally to the field of methods for conducting a chronic myelogenous leukemia (CML) assay to detect or monitor CML cells in a human patient.
  • CML chronic myelogenous leukemia
  • Chronic myelogenous leukemia is a hematological disorder that is the result of neoplastic transformation of pluripotent stem cells. Symptoms include fatigue, anemia, leukocytosis and splenomegaly.
  • the first phase is called the chronic phase and is associated with an increased production of committed myeloid and lymphoid progenitor cells. Terminal differentiation is maintained in this phase and an elevated granulocyte count is produced.
  • the chronic phase progresses to the second phase, which is also called the blast crisis phase. In the second phase, terminal differentiation is lost and unregulated proliferation of immature lymphoid and myeloid blast cells occurs.
  • the Philadelphia (Ph) chromosome was first described in 1960 as an abbreviated chromosome found in the bone marrow of patients with CML.
  • the Ph chromosome is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;qll).
  • the potential breakpoints on chromosome 22 band qll occur in a small 5.8 kb region called the breakpoint cluster region (ber).
  • the breakpoint cluster region is part of a large ber gene which contains four exons.
  • Potential breakpoints on chromosome 9 are scattered over a distance of at least 100 kb, but are all located 5' to the c-abl proto-oncogene.
  • the Ph translocation transfers the c-abl gene from its position on chromosome 9 band q34 to the Ph chromosome.
  • FIGURE 1 is a schematic drawing of the translocation region of the Philadelphia chromosome consisting of either bcrl-abll or bcr3-abl2.
  • FIGURE 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5.
  • the chimeric DNA produces a hybrid transcript containing either ber exon 2 to c-abl exon 2 or ber exon 3 to c-abl exon 2.
  • the resultant chimeric gene transcribes an 8.5 kb mRNA that contains 5' ber and 3' c-abl in a head-to-tail tandem sequence.
  • This hybrid protein encoded by the chimeric mRNA has elevated tyrosine kinase activity and its appearance parallels the onset of clinical symptoms of CML and ALL.
  • ALL the translocations of c-abl to ber have been mapped to the intron between exons pl and p2 of the ber gene.
  • This gene transcribes a 7.0 kb mRNA that also contains 5' ber and 3' c-abl in a head-to tail tandem sequence.
  • Current methods for diagnosing CML include karyotype determination and Southern blot analysis. Karyotype analysis requires expertise in cytogenetics and takes weeks to obtain the results. Southern blot analysis normally takes several days to perform and requires extensive laboratory equipment.
  • the present invention includes a method of detecting or monitoring human chronic myelogenous leukemia (CML) cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA.
  • CML chronic myelogenous leukemia
  • RNA from hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • RNA/capture agent complex comprising a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions is hybridized to the amplified RNA to form an RNA/capture agent complex.
  • This binding may be carried out at about 42°C.
  • Oligonucleotides specific for a target region are defined as oligonucleotides that hybridize specifically to the appropriate target under appropriate hybridization conditions.
  • the RNA/capture agent complex is coupled to a solid phase having a receptor specific for the binding ligand coupled to the capture agent. The solid phase is washed and a detector agent is added to the solid phase.
  • the detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions.
  • the addition of the detector agent may be carried out at room temperature.
  • the amount of the labeled detector oligonucleotide bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention also includes a method for detecting or monitoring CML cells in a human patient wherein a capture agent and a detector agent are simultaneously added to RNA that has been amplified using a pair of primers capable of amplifying both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the capture agent comprises a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions.
  • the target complex made up of the amplified RNA, bound capture agent and bound detector agent is then applied to a solid phase having a receptor specific for the binding ligand coupled to it.
  • the solid phase is then washed and the amount of detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention further includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA.
  • the RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions, and the amplification product is applied to a solid phase having a capture agent specific for the bcr2-abl2 and bcr3-abl2 translocation regions coupled to it.
  • the solid phase to which the amplified RNA is bound is then washed.
  • a detector agent comprising a detector label and an oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions is applied to the washed solid phase.
  • the amount of the detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
  • the present invention also includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal
  • RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the amplification product is then incubated with a labeled detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions to form a reaction product/ detector complex.
  • the reaction product/ detector complex is applied to a solid phase having a capture agent coupled to it, where the capture agent is specific for the bcr2-abl2 and bcr3-abl2 translocation regions.
  • the primer pairs used in the methods of the present invention may comprise a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site.
  • a sense-RNA oligonucleotide is a primer that generates a sense RNA in the amplification reaction and an antisense-RNA oligonucleotide is a primer that generates an antisense RNA in the amplification reaction.
  • These primers may be BB323/BB329, BB326/BB313 or BB325/BB329.
  • the RNA-based nucleic acid amplification may be self sustained sequence replication (3SR).
  • the binding ligand may be biotin and the receptor specific for the binding ligand may be either avidin or streptavidin.
  • the capture oligonucleotide may be CAP6, CAP7, CAP8, CAP10, CAP14 or CAP14T.
  • the detector oligonucleotide may be DET1, DET350, DET351, DET352, DET1-46 or DET1-3 and the detector label may be horseradish peroxidase, fluorescein, alkaline phosphatase or other appropriate detector molecule.
  • the solid phase may be a microwell plate, plastic beads, glass beads, magnetic particles or other solid phase materials known to those of ordinary skill in the diagnostic arts.
  • FIG. 1 is a schematic drawing of the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome.
  • Fig. 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5.
  • Fig. 3 depicts the hybridization regions for capture agents CAP6, CAP7, CAP8, CAP10 and CAP14.
  • Fig. 4 depicts the hybridization regions for detector agents DET350, DET351, DET352, DET618, DET643 and DET646 (DET646 is also called DET1).
  • Fig. 5 depicts the location of the regions of hybridization for the primer pairs BB170/BB332 and BB170/BB333.
  • Fig. 6 depicts the location of the regions of hybridization for the primer pairs BB172/BB173, BB172/BB174, BB172/BB175 and BB172/BB176.
  • Fig. 7 depicts the location of the regions of hybridization for the primer pairs BB189/BB329, BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 and BB189/BB188.
  • Fig. 8 depicts the location of the regions of hybridization for the primer pairs BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176 and BB316/BB309.
  • Fig. 9 depicts the location of the regions of hybridization for the primer pairs BB300/BB320, BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175 and BB300/BB176; BB300b/BB320, BB300b/BB173, BB300b/BB174, BB300b/BB175 and BB300b/BB176.
  • BB300 and BB300b hybridize to the same sequence region;
  • BB300 differs from BB300b in that it has a polymerase binding site sequence attached to it.
  • Fig. 10 depicts the location of the regions of hybridization for the primer pairs BB322/BB329 and BB322/BB331; BB323/BB329, BB323/BB330,
  • BB323/BB331 and BB323/BB333 BB325/BB313, BB325/BB328, BB325/BB329, BB325/BB330, BB325/BB331 and BB325/BB333; BB326/BB313, BB326/BB329 and BB326/BB330; and BB334/BB332 and BB334/BB333.
  • Fig. 11 depicts the hybridization regions for primer pair BB325/BB329, capture agent CAP14T and detector agent DET1.
  • Fig. 12 is a slot blot of 3SR products probed with oligo BB302, which is specific for the bcr2-abl2 translocation.
  • Fig. 13 is a slot blot of 3SR products probed with BB303, which is specific for the bcr3-abl2 translocation.
  • Fig. 14 is a summary of a portion of a clinical test. Karyotype results are given as the fraction of cells containing the Ph chromosome out of the total number of cells observed. Abbreviations are as follows: hydroxy urea (HU), interferon (IFN), true positive (TP), true negative (TN), false negative (FN). The CML EPA was scored as positive if the specimen absorbance value was greater than the positive cutoff value of 0.438 (i.e. 0.200 + OD value of the amplification blank).
  • the assays detect both the bcr2-abl2 and the bcr3-abl2 translocation associated with CML. These assays do not detect CML in the absence of the Ph chromosome, nor do they detect ALL even if the ALL patient has the Ph chromosome.
  • a blood sample is obtained from the CML patient and total RNA is extracted from the peripheral blood cells. The extracted RNA is contacted with primers that are capable of hybridizing at positions corresponding to sequences flanking the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome. The RNA is then subjected to an amplification procedure such as self-sustained sequence replication (3SR) or nucleic acid sequence-based amplification (NASBA).
  • 3SR self-sustained sequence replication
  • NASBA nucleic acid sequence-based amplification
  • the reaction product if any, is captured onto a solid phase such as a microwell by means of a capture agent.
  • a solid phase such as a microwell by means of a capture agent.
  • a 96-well microwell plate facilitates automation, as these plates can be read with an ELISA plate reader, a fluorogenic reader, or any other reader capable of detecting the selected detector agent. Plates other than 96-well plates may be used such as 6, 24 or 48 cluster plates. These plates are available in the form of strip wells or as conventional culture plates.
  • Other suitable solid phases include, without limitation, glass, plastic or magnetic beads or other acceptable surfaces.
  • the detector agent can be an oligonucleotide probe to which a label, such as horseradish peroxidase (HRP), has been coupled.
  • a solution-based hybridization can be performed.
  • the amplified sample is simultaneously mixed with a capture agent and a detector agent to form a target/capture agent/ detector agent complex.
  • the complex is then applied to a microwell.
  • the amount of label bound to the solid phase is measured and correlated with the amount of amplification.
  • the readings are analyzed to determine the presence, progression or regression of CML in a semi- quantitative manner.
  • the amplified sample is placed in a microwell plate coated with a capture agent. After the amplified sample is coupled to the bound capture agent, any unbound material is rinsed away. A detector agent is then applied under conditions that allow it to hybridize to the captured target, and the amount of label bound to the solid phase is measured.
  • the amplified sample can be incubated with the detector agent to form a sample /detector agent complex. The complex is then added to a microwell coated with a capture agent.
  • a microwell format is used to detect specific sequences derived from the bcr-abl chimeric mRNA. Transcripts are amplified using the self sustained sequence replication (3SR) reaction.
  • the assay comprises two fundamental steps. The first step involves isolation and 3SR amplification of bcr-abl mRNA from leukemic cells. The second step involves the capture and detection of the amplified product in a microwell.
  • the amplified sample is mixed with a biotinylated oligonucleotide that has a base sequence complementary to the abl region of the amplified bcr-abl product.
  • a biotinylated oligonucleotide that has a base sequence complementary to the abl region of the amplified bcr-abl product.
  • the sample is then added to a streptavidin coated microwell.
  • the biotin portion of the oligonucleotide binds with the streptavidin, thereby immobilizing any biotinylated capture oligonucleotides that are hybridized to the amplified bcr-abl product. Unbound products are subsequently washed out of the microwell.
  • Detection of the captured bcr-abl product is achieved with a peroxidase-labelled oligonucleotide that hybridizes to a sequence complementary to the ber region of the amplified product. Following the incubation, unhybridized peroxidase-labelled oligonucleotide is washed away. Substrate is added and color is produced if the capture- bcr- flfo/-peroxidase complex is present in the microwell. The color production is quantified and compared to negative and positive controls to determine the presence of bcr-abl RNA.
  • EXAMPLE 1 Oligonucleotides All oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer (Millipore, Marlborough, MA) using standard phosphoramidite chemistries as indicated by the manufacturer's directions. Oligonucleotides that contained a biotin molecule at the 5' end of the DNA were synthesized using biotin phosphoramidite (Glenn Research, Sterling, VA). Oligonucleotides that contained an amine at the 3' end of the DNA were made using an Amino-On column (catalogue number 20-2957-42, Glenn Research).
  • the primers that were tested for use in 3SR amplifications of the bcr2-abl2 and bcr3-abl2 translocation regions of the Philadelphia chromosome in the assay of the present invention are listed in Table 1 below and are identified in the Sequence Listing as SEQ. ID. Nos. 1-30 and 44. All primers listed below are oriented in the 5' to 3' direction from left to right.
  • sequence AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-A represents the T7 polymerase binding site with the final four bases (GGGA) being the preferred transcription start site.
  • Capture agents were oligonucleotide sequences coupled to a binding ligand, such as biotin. Capture agents that were tested in the present experiments are listed in Table 2 and are identified in the Sequence Listing as SEQ. ID. Nos. 31-36.
  • FIGURE 3 depicts the locations of the regions of hybridization for the capture agents. These capture oligonucleotide sequences were immobilized on streptavidin-coated plates as described in Example 5 below.
  • Detector agents were oligonucleotide sequences coupled to a label such as horseradish peroxidase (HRP), fluorescein, or alkaline phosphatase. Detector oligonucleotide sequences (amine-derivatized) that were tested in the present experiments are listed in Table 3 below and are identified in the Sequence Listing as SEQ. ID. Nos. 37-42.
  • FIGURE 4 depicts the location of the regions of hybridization for the capture agents.
  • K562 is a line of pleuroeffusion cells derived from a 53 year old woman suffering from chronic myelogenous leukemia (CML) in blast crisis.
  • CML chronic myelogenous leukemia
  • the K562 cell line contains the bcr2-abl2 translocation.
  • the cells were grown in RPMI with 10% fetal bovine serum and maintained by standard culture methods.
  • K562 cells were removed from culture vessels and then collected by centrifugation following the methods of Shtivelman, et al., Nature, 315: 550-554 (1985).
  • the cells were solubilized by vortexing at room temperature in 450 ⁇ l of lysing buffer comprising 0.5% SDS, 50 mM Tris pH 7.4, 0.1 M dithiothreitol, and 5 mM vanadyl ribonucleoside complex.
  • An equal volume of the salt solution, 1.6 M KCl and 50 mM MgCl 2 was added to each sample and detergent-protein complexes removed by centrifugation. The supernatant was precipitated with isopropanol or an equal volume of 5% w/v cetyltrimethylammonium bromide (CTAB).
  • CTCAB cetyltrimethylammonium bromide
  • the proteins can be extracted by using standard acid phenol extraction procedures.
  • the nucleic acids were collected by centrifugation and washed twice with 70% ethanol.
  • the various oligonucleotide primers were synthesized both with and without the T7 polymerase binding site and the amplification levels were determined. Surprisingly, it was found that the bcr-abl target, unlike most other targets, was amplified to higher levels when the polymerase binding site was on the primer that generated the sense RNA. All the primers set forth in Table 1 hybridized to total RNA isolated from the K562 cell line and functioned in amplification of the translocation region. FIGURES 5-10 depict the location of the regions of hybridization for the primers.
  • the preferred primer concentration for 3SR was generally about 15 pmole of each primer per reaction.
  • the preferred primer pairs were BB323/BB329, BB326/BB313 and BB325/BB329. The most preferred primer pair was BB325/BB329.
  • Primer pairs were tested in 3SR reaction mixes with and without dimethyl sulfoxide (DMSP) and sorbitol as additives for amplification.
  • DMSP dimethyl sulfoxide
  • a 5 ⁇ l aliquot of each nucleic acid sample was used as a template in a 3SR amplification reaction in an RNase-free 1.5 ml microcentrifuge tube.
  • the reaction mixture contained 20 ⁇ l of a 5X buffer (200 mM Tris HCl, pH 8.1, 150 mM MgCl 2 , 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine), 5 ⁇ l (15 pmol) of each of the priming oligonucleotides, 20 ⁇ l of a 5X nucleoside triphosphate mix (35 mM rNTP's, 5 mM dNTP's), and 45 ⁇ l of DEPC-treated H 2 P.
  • a 5X buffer 200 mM Tris HCl, pH 8.1, 150 mM MgCl 2 , 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine
  • 5 ⁇ l 15 pmol
  • BB189/BB329 BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176
  • BB300/BB320 BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175, BB300/BB176
  • BB300b/BB320 BB300b/BB173, BB300b/BB174, BB300b/BB175, BB300b/BB176
  • Assay Plate Design Micro wells were coated with streptavidin as follows. A solution containing 10 ⁇ g/ml streptavidin in 50 mM sodium borate, pH 9.0 was incubated in a covered microwell plate at 37°C for four hours. The plates were then washed five times with 0.05% Tween-20, 10 mM Tris, pH 7.4. The coated plates were then packed with a desiccant in an aluminum foil pouch and stored at 4°C until ready for use.
  • CAP14, CAP14T and CAP8 were the preferred capturing agents, giving the highest OD values at an absorbance wavelength of 450 nm (A 450 ) using a plate reader system sold by Baxter Healthcare
  • the concentration of the capture agent during in-solution hybridization with target also was an important factor in optimizing the assay. Excessive levels of capture agent inhibited specific binding to the streptavidin microwell.
  • Table 6 gives the signal-to-noise ratio of different concentrations of capture agents tested in the assay. The signal-to-noise ratio was calculated by comparing the OD (A 450 ) of wells containing target, detector agent and various concentrations of capture agent to wells containing only a labelled capture agent.
  • Optimal (highest) signals from capture agent CAP14T were obtained at 42°C.
  • Kinetics of hybridization of capture agents to the 3SR amplified target were optimized at 30 minutes in 0.1% polyvinylpyrrolidone (PVP), 2X SSC (0.3 M NaCl, 0.03 M sodium citrate).
  • the CML detection assay can be performed in a format in which the amplified target is incubated with the capture agent and the detector agent simultaneously in the microwell.
  • the amplified target sequence first is incubated with the capture agent to allow the capture agent to bind the target sequence.
  • the capture agent/target complex is applied to the microwells and unbound material is washed away.
  • the detector agent is applied to the microwell. The preferred procedure allows the detector agent to hybridize only to the captured 3SR target without the dilution effect seen in the simultaneous format, where detector agent can hybridize to free capture oligonucleotides in solution.
  • the preferred basic hybridization solution contained 0.1% PVP and 2X SSC.
  • the long term stability of the detector agent was also evaluated. It was found that by adding 1% (w/v) BSA and 30% (v/v) glycerol to the basic hybridization solution (0.1% PVP, 2X SSC), the life span of the detector agent was increased. The presence of glycerol had the added benefit of decreasing the background signal. A final concentration of 30% glycerol gave the maximum signal with the minimum background.
  • Figure 11 depicts the hybridization regions for the preferred primer pair BB325/BB329, preferred capture agent CAP14T and preferred detector agent DET1.
  • EXAMPLE 6 Isolation of Total RNA from Clinical Samples Three-milliliter blood samples were collected from patients and placed into citrate or EDTA treated tubes. Heparinized tubes were not used as they were found to inhibit the RNA amplification reaction. Samples were stored at room temperature and processed within 24 hours of specimen collection.
  • the tube containing the blood sample was mixed by inversion 5 times.
  • the entire 3 ml of blood was transferred into a 15 ml polypropylene conical tube.
  • Three milliliters of phosphate buffered saline (PBS) were added and mixed with the blood by inversion.
  • a Histopaque gradient was prepared by pipetting 6 ml Histopaque 1119 (Sigma Co., St. Louis, MO) into a 15 ml polypropylene conical tube.
  • Six milliliters of the diluted blood were slowly pipetted over the Histopaque, avoiding mixing.
  • the tube was placed into a clinical centrifuge (swinging bucket type) and spun at 900 x g for 30 minutes at room temperature.
  • the white blood cells (WBCs) from the serum/Histopaque interface were collected using a plugged pasteur pipette and transferred to another 15 ml sterile conical tube. The WBCs were counted using a hemocytometer. After counting the cells, 7 x IO 6 WBCs were transferred to another tube and the volume was raised to 10 mis with PBS. The cells were mixed by inversion and pelleted by centrifugation at 900 x g for 15 minutes at room temperature. The PBS was carefully decanted without disturbing the cell pellet. Another 10 ml of PBS was added, and the cells were spun at 900 x g for 15 minutes at room temperature. Again, the PBS was carefully decanted without disturbing the cell pellet.
  • RNAzol BTM (Teltest, Friendswood, TX) was added to the cell pellet and vortexed for 30 seconds or until the cell pellet was homogenized. Alternatively, the cell pellet was homogenized by repeatedly pipetting the solution. The homogenate was transferred to a sterile RNAse- free microcentrifuge tube. One hundred microliters of chloroform were added and the tube was vortexed for 30 seconds, stored on ice for 5 minutes and then centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous phase was carefully collected, avoiding the interphase, and transferred to another RNAse-free microcentrifuge tube.
  • RNA pellet was washed twice by adding 1.0 ml of 75% ethanol, vortexing for 5 seconds and centrifuging at 7,500 rpm for 5 minutes at 2-25 °C.
  • RNA pellet was stored on ice until used in the amplification process.
  • RNAse-free microcentrifuge tubes were labeled with the proper specimen or control number for the enzyme probe assay (EPA).
  • One positive amplification control and one amplification blank were run each time an amplification was performed.
  • the reagents in Module 1 i.e. amplification reagent, positive amplification control template, enzyme mix, capture buffer and detection reagent
  • All the reagents except the amplification reagent were then placed on ice.
  • the amplification reagent was warmed to 42°C. Once a reagent was used, it was returned to -70°C.
  • Reagent A amplification reagent composed of buffer (40 mM Tris HCl, pH 8.1, 30 mM MgCl 2 , 20 mM KCl, 10 mM dithiothreitol, 4 mM spermidine), 7 mM rNTP's, 1 mM dNTP's, and 15 pmol of each of the priming oligonucleotides BB325 and BB329) and 5 ⁇ l of Reagent B (positive amplification control template composed of 0.2 attamole/ ⁇ l in vitro generated RNA transcript of the bcr-abl region in 10 mM Tris, 1 mM EDTA, pH 7.4 ) were added to the microcentrifuge tube labelled "positive amplification control.”
  • Reagent A were added to the microcentrifuge tube labelled "amplification blank.” No template was added to the amplification blank.
  • Reamplification reagent A amplification reagent composed of
  • the tubes containing the specimens and controls were then transferred to a 65°C (+ 1°C) water bath for 2 minutes and then to a 41 °C ( ⁇ 1°C) water bath for 5 minutes.
  • Five microliters of Reagent C (enzyme mix containing 30 units AMV Reverse Transcriptase, 2 units RNAse H and 100 units T7 RNA polymerase) were added to each tube and the mixture was gently vortexed. The tube was observed to ensure that the RNA pellet was not attached to the upper walls of the microcentrifuge tube.
  • the mixture was incubated in a 41 °C ( ⁇ 1°C) water bath for 1 hour.
  • the number of EPA microwells needed for the assay was determined.
  • One negative control (amplification blank), two positive controls (one positive amplification control and one positive hybridization control), and one substrate control (empty well) were run each time the assay was performed.
  • the number of wells required for specimens and controls was determined.
  • An appropriate number of commercially available strips of pre-coated wells were removed from the packaging and placed in a 96-well frame provided by the manufacturer. Care was taken so as to not scratch the bottom surface of the wells as scratches could interfere with the reading of the test results.
  • Reagent D capture buffer composed of 8 pmol biotinylated oligonucleotide in 4X SSC, 0.2% PVP
  • Reagent D capture buffer composed of 8 pmol biotinylated oligonucleotide in 4X SSC, 0.2% PVP
  • microwell plate was covered with a plate lid and incubated at room temperature for 20 minutes. After the incubation, the plate lid was removed and each well was washed 3 times with 300 ⁇ l wash solution (2X SSC, 0.05% Tween-20, 0.01% thimerosal) using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 2 (detection reagent composed of 1 pmol peroxidase labelled detector oligonucleotide in 30% glycerol, 5X SSC, 0.1% PVP, and 1% BSA) was promptly added to the center of each well except the empty substrate control well.
  • Reagent 2 detection reagent composed of 1 pmol peroxidase labelled detector oligonucleotide in 30% glycerol, 5X SSC, 0.1% PVP, and 1% BSA
  • microwell plate was covered with the lid and incubated at room temperature for 30 minutes. Again the plate lid was removed and each well washed 3 times with 300 ⁇ l wash solution using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 3 (peroxidase substrate containing 0.04% (w/v) 3,3',5,5' tetramethylbenzidine
  • TMB Tetrahydrofurane
  • 0.02% hydrogen peroxide solution manufactured by Kirkegaard & Perry, Gaithersburg, MD
  • the microwell plate was covered with the plate lid and incubated at room temperature for 15 ⁇ 2 minutes.
  • the plate reader e.g. Baxter PRIMA or
  • Figure 14 summarizes additional clinical test results in which the results using the CML EPA can be compared to results using other tests.
  • the data in Figure 14 reflect a sensitivity of 91% and a specificity of 100%.
  • Sensitivity is defined as the proportion of people who truly have a specific disease and are so identified by a test.
  • Specificity is defined as the proportion of people who are truly free of a specific disease and are so identified by a test.
  • OD correlate with what would be expected of a valid clinical assay for CML. This test can also be used prognostically to assess cytogenetic remission in patients with chronic myelogenous leukemia.
  • the filters were pre-wetted with hybridization solution (6X SSC, 10X Denhardts, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS) and then hybridized at 68°C for 45 minutes with a 32p. labeled oligonucleotide probe that was complementary to the junction sequence of bcr2-abl2 (BB302) or bcr3-abl2 (BB303).
  • hybridization solution 6X SSC, 10X Denhardts, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS
  • the filters were washed three times at room temperature for 5 minutes each using 1 ml buffer /cm 2 filter, where the buffer was 2X SSC, 0. 1% SDS.
  • the filters were exposed to X-ray film at -70°C with one intensifying screen.
  • the results of the 3SR amplification using clinical specimens are shown in Figure 12 and 13.
  • nucleic acid specific for the bcr-abl translocation and isolated from clinical specimens can be amplified and detected. Further, it can be distinguished whether the cells contain the bcr2-abl2 or the bcr3-abl2 translocation.

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Abstract

Methods are provided for conducting chronic myelogenous leukemia (CML) assays to detect or monitor CML cells in a human patient. A sample is obtained from the patient and total RNA is extracted. The extracted RNA is contacted with appropriate primers that surround the bcr2-abl2 or the bcr3-abl2 translocation regions of the Philadelphia chromosome, and the inter-primer regions are amplified. After amplification, the reaction product, if any, is captured onto a solid phase by means of a capture agent and is detected by means of a labelled detector agent. The amount of labelled detector agent is correlated with the presence or quantity of CML cells in the patient.

Description

CHRONIC MYELOGENOUS LEUKEMIA DIAGNOSTIC ASSAY
FIELD OF THE INVENTION This invention relates generally to the field of methods for conducting a chronic myelogenous leukemia (CML) assay to detect or monitor CML cells in a human patient.
BACKGROUND OF THE INVENTION Chronic myelogenous leukemia (CML) is a hematological disorder that is the result of neoplastic transformation of pluripotent stem cells. Symptoms include fatigue, anemia, leukocytosis and splenomegaly. There are two clinical phases that exemplify CML. The first phase is called the chronic phase and is associated with an increased production of committed myeloid and lymphoid progenitor cells. Terminal differentiation is maintained in this phase and an elevated granulocyte count is produced. The chronic phase progresses to the second phase, which is also called the blast crisis phase. In the second phase, terminal differentiation is lost and unregulated proliferation of immature lymphoid and myeloid blast cells occurs. The Philadelphia (Ph) chromosome was first described in 1960 as an abbreviated chromosome found in the bone marrow of patients with CML. The Ph chromosome is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;qll). The potential breakpoints on chromosome 22 band qll occur in a small 5.8 kb region called the breakpoint cluster region (ber). The breakpoint cluster region is part of a large ber gene which contains four exons. Potential breakpoints on chromosome 9 are scattered over a distance of at least 100 kb, but are all located 5' to the c-abl proto-oncogene. The Ph translocation transfers the c-abl gene from its position on chromosome 9 band q34 to the Ph chromosome.
Because 90% of CML patients carry the Ph chromosome, it constitutes the hallmark of CML and is diagnostic of the disease. Approximately 5% of the childhood and 30% of the adult acute lymphocytic leukemias (ALL) also carry the Ph chromosome. The Ph chromosome in CML and ALL results from the same translocation of c-abl to different introns of the ber gene. In CML, the translocations of c-abl to ber have been mapped to two regions, between ber exons 2 and 3 or between ber exons 3 and 4. FIGURE 1 is a schematic drawing of the translocation region of the Philadelphia chromosome consisting of either bcrl-abll or bcr3-abl2. FIGURE 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5. The chimeric DNA produces a hybrid transcript containing either ber exon 2 to c-abl exon 2 or ber exon 3 to c-abl exon 2. Clinically there is no difference between the bcr2-abl2 and the bcr3-abl2 translocations even though they occur in different areas of the chromosome. In either case, the resultant chimeric gene transcribes an 8.5 kb mRNA that contains 5' ber and 3' c-abl in a head-to-tail tandem sequence. This hybrid protein encoded by the chimeric mRNA has elevated tyrosine kinase activity and its appearance parallels the onset of clinical symptoms of CML and ALL. In ALL, the translocations of c-abl to ber have been mapped to the intron between exons pl and p2 of the ber gene. This gene transcribes a 7.0 kb mRNA that also contains 5' ber and 3' c-abl in a head-to tail tandem sequence. Current methods for diagnosing CML include karyotype determination and Southern blot analysis. Karyotype analysis requires expertise in cytogenetics and takes weeks to obtain the results. Southern blot analysis normally takes several days to perform and requires extensive laboratory equipment. These techniques have limited sensitivity because they can only detect disease states in which at least 5% of the white blood cells possess the Ph chromosome. Many times cytogenetic analysis produces sub¬ standard results because an inadequate number of cells were collected or because the cells that were collected were not viable. This makes an adequate assessment of remission difficult using cytogenetic analysis.
SUMMARY OF THE INVENTION The present invention includes a method of detecting or monitoring human chronic myelogenous leukemia (CML) cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA. RNA from hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions. Next, a capture agent comprising a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions is hybridized to the amplified RNA to form an RNA/capture agent complex. This binding may be carried out at about 42°C. Oligonucleotides specific for a target region are defined as oligonucleotides that hybridize specifically to the appropriate target under appropriate hybridization conditions. The RNA/capture agent complex is coupled to a solid phase having a receptor specific for the binding ligand coupled to the capture agent. The solid phase is washed and a detector agent is added to the solid phase. The detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions. The addition of the detector agent may be carried out at room temperature. Last, the amount of the labeled detector oligonucleotide bound to the solid phase is correlated with the presence or quantity of CML cells in the patient. - A -
The present invention also includes a method for detecting or monitoring CML cells in a human patient wherein a capture agent and a detector agent are simultaneously added to RNA that has been amplified using a pair of primers capable of amplifying both the bcr2-abl2 and bcr3-abl2 translocation regions. The capture agent comprises a binding ligand and a capture oligonucleotide specific for the bcr2-abl2 and bcr3-abl2 translocation regions. The detector agent comprises a detector label and a detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions. The target complex made up of the amplified RNA, bound capture agent and bound detector agent is then applied to a solid phase having a receptor specific for the binding ligand coupled to it. The solid phase is then washed and the amount of detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
The present invention further includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA. The RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions, and the amplification product is applied to a solid phase having a capture agent specific for the bcr2-abl2 and bcr3-abl2 translocation regions coupled to it. The solid phase to which the amplified RNA is bound is then washed. Subsequently, a detector agent comprising a detector label and an oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions is applied to the washed solid phase. Last, the amount of the detector agent bound to the solid phase is correlated with the presence or quantity of CML cells in the patient.
The present invention also includes the following method for detecting or monitoring human CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal
DNA. The RNA from a patient's hematopoietic cells is amplified by means of an RNA-based nucleic acid amplification procedure using a pair of primers capable of causing amplification of both the bcr2-abl2 and bcr3-abl2 translocation regions. The amplification product is then incubated with a labeled detector oligonucleotide specific for the bcr2-abl2 or bcr3-abl2 translocation regions to form a reaction product/ detector complex. The reaction product/ detector complex is applied to a solid phase having a capture agent coupled to it, where the capture agent is specific for the bcr2-abl2 and bcr3-abl2 translocation regions. The solid phase is washed and the amount of labeled detector oligonucleotide bound to the solid phase is correlated with the presence or quantity of CML cells in the patient. The primer pairs used in the methods of the present invention may comprise a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site. A sense-RNA oligonucleotide is a primer that generates a sense RNA in the amplification reaction and an antisense-RNA oligonucleotide is a primer that generates an antisense RNA in the amplification reaction. These primers may be BB323/BB329, BB326/BB313 or BB325/BB329. The RNA-based nucleic acid amplification may be self sustained sequence replication (3SR).
The binding ligand may be biotin and the receptor specific for the binding ligand may be either avidin or streptavidin. The capture oligonucleotide may be CAP6, CAP7, CAP8, CAP10, CAP14 or CAP14T. The detector oligonucleotide may be DET1, DET350, DET351, DET352, DET1-46 or DET1-3 and the detector label may be horseradish peroxidase, fluorescein, alkaline phosphatase or other appropriate detector molecule. The solid phase may be a microwell plate, plastic beads, glass beads, magnetic particles or other solid phase materials known to those of ordinary skill in the diagnostic arts.
BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a schematic drawing of the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome. Fig. 2 gives the DNA sequence for the human chimeric bcr/c-abl fusion protein gene, exons 2-5. Fig. 3 depicts the hybridization regions for capture agents CAP6, CAP7, CAP8, CAP10 and CAP14.
Fig. 4 depicts the hybridization regions for detector agents DET350, DET351, DET352, DET618, DET643 and DET646 (DET646 is also called DET1).
Fig. 5 depicts the location of the regions of hybridization for the primer pairs BB170/BB332 and BB170/BB333.
Fig. 6 depicts the location of the regions of hybridization for the primer pairs BB172/BB173, BB172/BB174, BB172/BB175 and BB172/BB176. Fig. 7 depicts the location of the regions of hybridization for the primer pairs BB189/BB329, BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 and BB189/BB188.
Fig. 8 depicts the location of the regions of hybridization for the primer pairs BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176 and BB316/BB309.
Fig. 9 depicts the location of the regions of hybridization for the primer pairs BB300/BB320, BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175 and BB300/BB176; BB300b/BB320, BB300b/BB173, BB300b/BB174, BB300b/BB175 and BB300b/BB176. BB300 and BB300b hybridize to the same sequence region; BB300 differs from BB300b in that it has a polymerase binding site sequence attached to it.
Fig. 10 depicts the location of the regions of hybridization for the primer pairs BB322/BB329 and BB322/BB331; BB323/BB329, BB323/BB330,
BB323/BB331 and BB323/BB333; BB325/BB313, BB325/BB328, BB325/BB329, BB325/BB330, BB325/BB331 and BB325/BB333; BB326/BB313, BB326/BB329 and BB326/BB330; and BB334/BB332 and BB334/BB333.
Fig. 11 depicts the hybridization regions for primer pair BB325/BB329, capture agent CAP14T and detector agent DET1.
Fig. 12 is a slot blot of 3SR products probed with oligo BB302, which is specific for the bcr2-abl2 translocation.
Fig. 13 is a slot blot of 3SR products probed with BB303, which is specific for the bcr3-abl2 translocation. Fig. 14 is a summary of a portion of a clinical test. Karyotype results are given as the fraction of cells containing the Ph chromosome out of the total number of cells observed. Abbreviations are as follows: hydroxy urea (HU), interferon (IFN), true positive (TP), true negative (TN), false negative (FN). The CML EPA was scored as positive if the specimen absorbance value was greater than the positive cutoff value of 0.438 (i.e. 0.200 + OD value of the amplification blank).
DETAILED DESCRIPTION OF THE INVENTION The assays of the present invention are used to detect or monitor
CML cells in a human patient. The assays detect both the bcr2-abl2 and the bcr3-abl2 translocation associated with CML. These assays do not detect CML in the absence of the Ph chromosome, nor do they detect ALL even if the ALL patient has the Ph chromosome. A blood sample is obtained from the CML patient and total RNA is extracted from the peripheral blood cells. The extracted RNA is contacted with primers that are capable of hybridizing at positions corresponding to sequences flanking the bcr2-abl2 and bcr3-abl2 translocation region of the Philadelphia chromosome. The RNA is then subjected to an amplification procedure such as self-sustained sequence replication (3SR) or nucleic acid sequence-based amplification (NASBA).
Guatelli et al., Isothermal In Vitro Amplification of Nucleic Acids by a Multi¬ enzyme Reaction Modified After Retroviral Replication, Proc. Nat'l Acad. Sci. USA 87:1874 (1990); Malek, L. et al., U.S. Patent No. 5,130,238. If the characteristic CML translocation has occurred in the patient, the amplification procedure will yield a reaction product encompassing the translocation region. If the translocation has not occurred in the patient, no amplification product will be formed.
After the amplification procedure, the reaction product, if any, is captured onto a solid phase such as a microwell by means of a capture agent. The use of a 96-well microwell plate facilitates automation, as these plates can be read with an ELISA plate reader, a fluorogenic reader, or any other reader capable of detecting the selected detector agent. Plates other than 96-well plates may be used such as 6, 24 or 48 cluster plates. These plates are available in the form of strip wells or as conventional culture plates. Other suitable solid phases include, without limitation, glass, plastic or magnetic beads or other acceptable surfaces. After the reaction product is coupled to the solid phase, any unbound material is rinsed away. A detector agent is then applied under conditions that allow it to hybridize to the captured target. The detector agent can be an oligonucleotide probe to which a label, such as horseradish peroxidase (HRP), has been coupled. Alternative to the above- described solid phase hybridization, a solution-based hybridization can be performed. In a solution-based hybridization, the amplified sample is simultaneously mixed with a capture agent and a detector agent to form a target/capture agent/ detector agent complex. The complex is then applied to a microwell. The amount of label bound to the solid phase is measured and correlated with the amount of amplification. The readings are analyzed to determine the presence, progression or regression of CML in a semi- quantitative manner.
In another embodiment, the amplified sample is placed in a microwell plate coated with a capture agent. After the amplified sample is coupled to the bound capture agent, any unbound material is rinsed away. A detector agent is then applied under conditions that allow it to hybridize to the captured target, and the amount of label bound to the solid phase is measured. Alternatively, the amplified sample can be incubated with the detector agent to form a sample /detector agent complex. The complex is then added to a microwell coated with a capture agent. In a preferred embodiment, a microwell format is used to detect specific sequences derived from the bcr-abl chimeric mRNA. Transcripts are amplified using the self sustained sequence replication (3SR) reaction. The assay comprises two fundamental steps. The first step involves isolation and 3SR amplification of bcr-abl mRNA from leukemic cells. The second step involves the capture and detection of the amplified product in a microwell.
To accomplish this second step, the amplified sample is mixed with a biotinylated oligonucleotide that has a base sequence complementary to the abl region of the amplified bcr-abl product. During incubation the biotinylated oligonucleotide specifically hybridizes to the abl region of the amplified product. The sample is then added to a streptavidin coated microwell. The biotin portion of the oligonucleotide binds with the streptavidin, thereby immobilizing any biotinylated capture oligonucleotides that are hybridized to the amplified bcr-abl product. Unbound products are subsequently washed out of the microwell. Detection of the captured bcr-abl product is achieved with a peroxidase-labelled oligonucleotide that hybridizes to a sequence complementary to the ber region of the amplified product. Following the incubation, unhybridized peroxidase-labelled oligonucleotide is washed away. Substrate is added and color is produced if the capture- bcr- flfo/-peroxidase complex is present in the microwell. The color production is quantified and compared to negative and positive controls to determine the presence of bcr-abl RNA. The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention as described in the claims. In the specific experiments described, Examples 1-5 cover optimization of the assay procedures using cultured cells known to carry the CML translocation. Examples 6-8 cover procedures and results for various clinical samples.
EXAMPLE 1 Oligonucleotides All oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer (Millipore, Marlborough, MA) using standard phosphoramidite chemistries as indicated by the manufacturer's directions. Oligonucleotides that contained a biotin molecule at the 5' end of the DNA were synthesized using biotin phosphoramidite (Glenn Research, Sterling, VA). Oligonucleotides that contained an amine at the 3' end of the DNA were made using an Amino-On column (catalogue number 20-2957-42, Glenn Research). The primers that were tested for use in 3SR amplifications of the bcr2-abl2 and bcr3-abl2 translocation regions of the Philadelphia chromosome in the assay of the present invention are listed in Table 1 below and are identified in the Sequence Listing as SEQ. ID. Nos. 1-30 and 44. All primers listed below are oriented in the 5' to 3' direction from left to right.
TABLE 1
SEP. ID. No. Primer Sequence*
1 BB161 GCA-GTT-TCG-GGC-AGC-AAG-AT
2 BB164 TCT-GAC-TAT-GAG-CGT-GCA-GAG
3 BB165 ACT-GCT-CTC-ACT-CTC-ACG-CA
BB170 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATC- GGA-GCA-GGA-GTC-ACT-GCT-GCT-GC
BB172 AAT-TTA-ATA-C GA-CTC-ACT-ATA-G GG-AAG- CCG-CAA-CGG-CAA-GAG-TTA-CAC
BB173 AAT-TTA-ATA-CG A-CTC-ACT-ATA-GGG-ACT-CCA- CTG-GCC-ACA-AAA-TCA-TAC-AG
BB174 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATA-TCT- CCA-CTG-GCC-ACA-AAA-TCA-TA
BB175 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATG-TTA- TCT-CCA-CTG-GCC-ACA-AAA-TC
BB176 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATT- AGA-GTG-TTA-TCT-CCA-CTG-GC
10 BB188 GGC-CAC-AAA-ATC-ATA-CAG-TGC
11 BB189 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ACT-TCA- GGG-TGC-ACA-GCC-GCA-ACG
12 BB300 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-AGG-
AGC-TGC-AGA-TGC-TGA-CCA-AC
13 BB300b GGA-GCT-GCA-GAT-GCT-GAC-CAA-C
14 BB302 GCT-GAA-GGG-CTT-TTG-AAC-TCT-GCT-TA
44 BB303 GCT-GAA-GGG-CTT-CTT-CCT-TAT-TGA-TG 15 BB304 TAA-GCA-GAG-TTC-AAA-AGC-CCT-TCA-GC
16 BB309 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATT-TTT-
GGT-TTG-GGC-TTC-ACA
17 BB313 AAT-TTA-ATA-C GA-CTC-ACT-ATA-GGG-AGG-
GGT-CAT-TTT-CAC-TGG-GTC
18 BB316 AAT-TTA-ATA-C GA-CTC-ACT-ATA-GGG-AAG- ACT-GTC-CAC-AGC-ATT-CCG
19 BB320b CAC-TGG-CCA-CAA-AAT-CAT-ACA-GTG-C
20 BB322 AAT-TTA-ATA-CGA-CTC-ACT-ATA-G GG-ATG-
CAG-ATG-CTG-ACC-AAC-TCG-TG
21 BB323 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-AAT-GCT- GAC-CAA-CTC-GTG-TGT-GA
22 BB325 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-AAG- ATG-CTG-ACC-AAC-TCG-TGT-GT
23 BB326 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-AGC-
TGA-CCA-ACT-CGT-GTG-TGA-AA
24 BB328 CGA-AAA-GGT-TGG-GGT-CAT-TT
25 BB329 TGC-AAC-GAA-AAG-GTT-GGG-GT
26 BB330 TAC-AGT-GCA-ACG-AAA-AGG-TT
27 BB331 AAT-CAT-ACA-GTG-CAA-CGA-AAA-G
28 BB332 CAA-AAT-CAT-ACA-GTG-CAA-CGA-A
29 BB333 CCA-CAA-AAT-CAT-ACA-GTG-CAA-C
30 BB334 AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-ATC- AGG-GTG-CAC-AGC-CGC-AAC-GGC
*The sequence AAT-TTA-ATA-CGA-CTC-ACT-ATA-GGG-A represents the T7 polymerase binding site with the final four bases (GGGA) being the preferred transcription start site.
Capture agents were oligonucleotide sequences coupled to a binding ligand, such as biotin. Capture agents that were tested in the present experiments are listed in Table 2 and are identified in the Sequence Listing as SEQ. ID. Nos. 31-36. FIGURE 3 depicts the locations of the regions of hybridization for the capture agents. These capture oligonucleotide sequences were immobilized on streptavidin-coated plates as described in Example 5 below.
TABLE 2
SEP. ID. No. Primer Sequence
31 CAP6 BIOTIN-TCA-CTG-GGT-CCA-G
32 CAP7 BIOTIN-TCC-TTG-GAG-TTC-CAA-CGA-G
33 CAP8 BIOTIN-AGC-GAG-AAG-GTT-TTC-CT 34 CAP10 BIOTIN-CGA-GCG-GCT-TCA-CTC-AG
35 CAP14 BIOTIN-GAT-GCT-ACT-GGC-CGC-TGA-AGG-GC
36 CAP14T BIOTIN-TTT-TTT-GAT-GCT-ACT-GGC-CGC-TGA- AGG-GC
Detector agents were oligonucleotide sequences coupled to a label such as horseradish peroxidase (HRP), fluorescein, or alkaline phosphatase. Detector oligonucleotide sequences (amine-derivatized) that were tested in the present experiments are listed in Table 3 below and are identified in the Sequence Listing as SEQ. ID. Nos. 37-42. FIGURE 4 depicts the location of the regions of hybridization for the capture agents.
TABLE 3
SEP. ID. No. Primer Sequence
37 DET1 CTT-CCT-TAT-TGA-TGG-TCA-NH2
38 DET350 GGT-CAG-CGG-AAT-GCT-GTG-GA-NH2
39 DET351 TCA-TCA-TCT-TCC-TTA-TTG-AT-NH2
40 DET352 ACA-TTC-AGA-AAC-CCA-TAG-AG- NH2 41 DET618 GTG-GAC-AGT-CTG-GAG-TTT- NH2
42 DET643 CCT-TAT-TGA-TGG-TCA-GCG-NH2
EXAMPLE 2
Isolation of Total RNA from Tissue Cultured Cells A sample of human K562 cultured cells was obtained from American Type Culture Collection (Rockville, Maryland). K562 is a line of pleuroeffusion cells derived from a 53 year old woman suffering from chronic myelogenous leukemia (CML) in blast crisis. The K562 cell line contains the bcr2-abl2 translocation. The cells were grown in RPMI with 10% fetal bovine serum and maintained by standard culture methods. K562 cells were removed from culture vessels and then collected by centrifugation following the methods of Shtivelman, et al., Nature, 315: 550-554 (1985). The cells were solubilized by vortexing at room temperature in 450 μl of lysing buffer comprising 0.5% SDS, 50 mM Tris pH 7.4, 0.1 M dithiothreitol, and 5 mM vanadyl ribonucleoside complex. An equal volume of the salt solution, 1.6 M KCl and 50 mM MgCl2, was added to each sample and detergent-protein complexes removed by centrifugation. The supernatant was precipitated with isopropanol or an equal volume of 5% w/v cetyltrimethylammonium bromide (CTAB). Proteins were extracted using a commercially available matrix such as Strataclean™ (Stratagene, La Jolla CA), Pro-Cipitate™ (Affinity Technology Inc., New Brunswick, NY), or other protein binding columns.
Alternatively, the proteins can be extracted by using standard acid phenol extraction procedures. The nucleic acids were collected by centrifugation and washed twice with 70% ethanol. These methods are set forth in U.S. Patent Application Serial No. 08/264,556 entitled "Rapid Isolation of Nucleic Acid" filed on June 23, 1994, incorporated by reference herein. EXAMPLE 3
Primer Pairs Used in Amplification
The various oligonucleotide primers were synthesized both with and without the T7 polymerase binding site and the amplification levels were determined. Surprisingly, it was found that the bcr-abl target, unlike most other targets, was amplified to higher levels when the polymerase binding site was on the primer that generated the sense RNA. All the primers set forth in Table 1 hybridized to total RNA isolated from the K562 cell line and functioned in amplification of the translocation region. FIGURES 5-10 depict the location of the regions of hybridization for the primers. The preferred primer concentration for 3SR was generally about 15 pmole of each primer per reaction. The preferred primer pairs were BB323/BB329, BB326/BB313 and BB325/BB329. The most preferred primer pair was BB325/BB329.
Primer pairs were tested in 3SR reaction mixes with and without dimethyl sulfoxide (DMSP) and sorbitol as additives for amplification. A 5 μl aliquot of each nucleic acid sample was used as a template in a 3SR amplification reaction in an RNase-free 1.5 ml microcentrifuge tube. The reaction mixture contained 20 μl of a 5X buffer (200 mM Tris HCl, pH 8.1, 150 mM MgCl2, 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine), 5μl (15 pmol) of each of the priming oligonucleotides, 20 μl of a 5X nucleoside triphosphate mix (35 mM rNTP's, 5 mM dNTP's), and 45 μl of DEPC-treated H2P. Various amounts of additive were also included in the reaction mixtures, i.e., 5%, 10%, 15% and 20% v/v for DMSP and 5%, 10%, 15% and 20% v/v for sorbitol. Each mixture was heat denatured at 65°C for 1 minute. Following denaturation, each tube was transferred to a 42° C water bath and incubated for 5 minutes. Thirty units of AMV reverse transcriptase, 2 units of E. coli Ribonuclease H and 1000 units of T7 RNA polymerase were added to each tube and the mixtures were incubated at 42°C for 60 minutes. The reaction product was analyzed by slot blot following the procedure of EXAMPLE 8 below. Table 4 lists primer pairs that were experimentally demonstrated to amplify both the bcr2-abl2 and the bcr3-abl2 translocation regions.
TABLE 4
Primer Pairs That Require 15% Sorbitol and 10% DMSO as Additives for Amplification:
BB170/BB332, BB170/BB333
BB172/BB173, BB172/BB174, BB172/BB175, BB172/BB176
BB189/BB329, BB189/BB330, BB189/BB331, BB189/BB332, BB189/BB333 BB316/BB173, BB316/BB174, BB316/BB175, BB316/BB176
BB316/BB309
Primer Pairs That Require No Additives for Amplification:
BB300/BB320, BB300/BB329, BB300/BB173, BB300/BB174, BB300/BB175, BB300/BB176
BB300b/BB320, BB300b/BB173, BB300b/BB174, BB300b/BB175, BB300b/BB176
BB322/BB329, BB322/BB331
BB323/BB329, BB323/BB330, BB323/BB331, BB323/BB333
BB325/BB313, BB325/BB328, BB325/BB329, BB325/BB330, BB325/BB331, BB325/BB333
BB326/BB313, BB326/BB329, BB326/BB330
BB334/BB332, BB334/BB333
EXAMPLE 4
Assay Plate Design Micro wells were coated with streptavidin as follows. A solution containing 10 μg/ml streptavidin in 50 mM sodium borate, pH 9.0 was incubated in a covered microwell plate at 37°C for four hours. The plates were then washed five times with 0.05% Tween-20, 10 mM Tris, pH 7.4. The coated plates were then packed with a desiccant in an aluminum foil pouch and stored at 4°C until ready for use.
EXAMPLE 5
Optimization of Assay Reagents and Design
Certain capture agents and detector agents worked more effectively than others. CAP14, CAP14T and CAP8 were the preferred capturing agents, giving the highest OD values at an absorbance wavelength of 450 nm (A450) using a plate reader system sold by Baxter Healthcare
Corporation (PRIMA System). Capture oligonucleotides of less than 17 nucleotides were not effective under the present assay conditions. Most differences in signals could be attributed to variations in optimum hybridization temperatures, implicating secondary structure as a limiting factor in these hybridizations.
The concentration of the capture agent during in-solution hybridization with target also was an important factor in optimizing the assay. Excessive levels of capture agent inhibited specific binding to the streptavidin microwell. Table 6 gives the signal-to-noise ratio of different concentrations of capture agents tested in the assay. The signal-to-noise ratio was calculated by comparing the OD (A450) of wells containing target, detector agent and various concentrations of capture agent to wells containing only a labelled capture agent.
Table 5
Signal to Noise Ratios for Different Concentrations of A Capture Agent
Capture agent concentration (pmole) signal: noise ratio
2.2/well 2.0 4.3/well 2.4
5.6/well 1.2
7.2/well 0.9
9.5/well 0.5 The inhibition of the specific binding of capture agent to streptavidin may have been due to "shielding," a charge interaction of the biotinylated DNA with the streptavidin. When 1 pmol of capture agent was applied to a well, approximately 40% of the capture agent bound; when 4 pmol of capture agent was applied, only about 25% bound. When the concentration of the capture agent was increased to over 10 pmol, very little binding occurred. In order to demonstrate that the inhibition was not due to excess DNA per se, a preparation of the biotinylated capture agent was spiked with unbiotinylated capture agent that had an identical nucleotide sequence and the preparation was used in a hybridization reaction. Results indicated that inhibition was not due to excess DNA per se. Optimal (highest) signals from capture agent CAP14T were obtained at 42°C. Kinetics of hybridization of capture agents to the 3SR amplified target were optimized at 30 minutes in 0.1% polyvinylpyrrolidone (PVP), 2X SSC (0.3 M NaCl, 0.03 M sodium citrate).
The CML detection assay can be performed in a format in which the amplified target is incubated with the capture agent and the detector agent simultaneously in the microwell. However, in a preferred embodiment, the amplified target sequence first is incubated with the capture agent to allow the capture agent to bind the target sequence. Next, the capture agent/target complex is applied to the microwells and unbound material is washed away. Finally, the detector agent is applied to the microwell. The preferred procedure allows the detector agent to hybridize only to the captured 3SR target without the dilution effect seen in the simultaneous format, where detector agent can hybridize to free capture oligonucleotides in solution.
Various hybridization solutions were tested. Most of the ingredients were found to have little or no effect on hybridization rates or background levels. The preferred basic hybridization solution contained 0.1% PVP and 2X SSC. The long term stability of the detector agent was also evaluated. It was found that by adding 1% (w/v) BSA and 30% (v/v) glycerol to the basic hybridization solution (0.1% PVP, 2X SSC), the life span of the detector agent was increased. The presence of glycerol had the added benefit of decreasing the background signal. A final concentration of 30% glycerol gave the maximum signal with the minimum background.
Figure 11 depicts the hybridization regions for the preferred primer pair BB325/BB329, preferred capture agent CAP14T and preferred detector agent DET1.
EXAMPLE 6 Isolation of Total RNA from Clinical Samples Three-milliliter blood samples were collected from patients and placed into citrate or EDTA treated tubes. Heparinized tubes were not used as they were found to inhibit the RNA amplification reaction. Samples were stored at room temperature and processed within 24 hours of specimen collection.
The tube containing the blood sample was mixed by inversion 5 times. The entire 3 ml of blood was transferred into a 15 ml polypropylene conical tube. Three milliliters of phosphate buffered saline (PBS) were added and mixed with the blood by inversion. A Histopaque gradient was prepared by pipetting 6 ml Histopaque 1119 (Sigma Co., St. Louis, MO) into a 15 ml polypropylene conical tube. Six milliliters of the diluted blood were slowly pipetted over the Histopaque, avoiding mixing. The tube was placed into a clinical centrifuge (swinging bucket type) and spun at 900 x g for 30 minutes at room temperature. The white blood cells (WBCs) from the serum/Histopaque interface were collected using a plugged pasteur pipette and transferred to another 15 ml sterile conical tube. The WBCs were counted using a hemocytometer. After counting the cells, 7 x IO6 WBCs were transferred to another tube and the volume was raised to 10 mis with PBS. The cells were mixed by inversion and pelleted by centrifugation at 900 x g for 15 minutes at room temperature. The PBS was carefully decanted without disturbing the cell pellet. Another 10 ml of PBS was added, and the cells were spun at 900 x g for 15 minutes at room temperature. Again, the PBS was carefully decanted without disturbing the cell pellet. One milliliter of RNAzol B™ (Teltest, Friendswood, TX) was added to the cell pellet and vortexed for 30 seconds or until the cell pellet was homogenized. Alternatively, the cell pellet was homogenized by repeatedly pipetting the solution. The homogenate was transferred to a sterile RNAse- free microcentrifuge tube. One hundred microliters of chloroform were added and the tube was vortexed for 30 seconds, stored on ice for 5 minutes and then centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous phase was carefully collected, avoiding the interphase, and transferred to another RNAse-free microcentrifuge tube.
One-half milliliter of 100% isopropanol was added, the tube was vortexed for 10 seconds and then stored on ice for 15 minutes. Alternatively, the sample can be stored at -20°C for up to 2 months. The sample was centrifuged at 12,000 x g for 15 minutes at 4°C, after which the isopropanol was carefully decanted away from the RNA pellet without disturbing the pellet. The RNA pellet was washed twice by adding 1.0 ml of 75% ethanol, vortexing for 5 seconds and centrifuging at 7,500 rpm for 5 minutes at 2-25 °C.
Following a brief centrifugation, the residual 75% ethanol was removed by pipetting. The RNA pellet was stored on ice until used in the amplification process.
EXAMPLE 7
Clinical CML Enzyme Probe Assay (EPA) Procedure RNAse-free microcentrifuge tubes were labeled with the proper specimen or control number for the enzyme probe assay (EPA). One positive amplification control and one amplification blank were run each time an amplification was performed. The reagents in Module 1 (i.e. amplification reagent, positive amplification control template, enzyme mix, capture buffer and detection reagent) were thawed. All the reagents except the amplification reagent were then placed on ice. The amplification reagent was warmed to 42°C. Once a reagent was used, it was returned to -70°C. One hundred microliters of Reagent A (amplification reagent composed of buffer (40 mM Tris HCl, pH 8.1, 30 mM MgCl2, 20 mM KCl, 10 mM dithiothreitol, 4 mM spermidine), 7 mM rNTP's, 1 mM dNTP's, and 15 pmol of each of the priming oligonucleotides BB325 and BB329) and 5 μl of Reagent B (positive amplification control template composed of 0.2 attamole/μl in vitro generated RNA transcript of the bcr-abl region in 10 mM Tris, 1 mM EDTA, pH 7.4 ) were added to the microcentrifuge tube labelled "positive amplification control." One hundred microliters of Reagent A were added to the microcentrifuge tube labelled "amplification blank." No template was added to the amplification blank. One hundred microliters of Reagent A were added to each microcentrifuge tube containing a specimen RNA pellet and the tube was vortexed briefly. All specimen and control tubes were kept on ice during this stage of the procedure.
The tubes containing the specimens and controls were then transferred to a 65°C (+ 1°C) water bath for 2 minutes and then to a 41 °C (± 1°C) water bath for 5 minutes. Five microliters of Reagent C (enzyme mix containing 30 units AMV Reverse Transcriptase, 2 units RNAse H and 100 units T7 RNA polymerase) were added to each tube and the mixture was gently vortexed. The tube was observed to ensure that the RNA pellet was not attached to the upper walls of the microcentrifuge tube. The mixture was incubated in a 41 °C (± 1°C) water bath for 1 hour.
The number of EPA microwells needed for the assay was determined. One negative control (amplification blank), two positive controls (one positive amplification control and one positive hybridization control), and one substrate control (empty well) were run each time the assay was performed. The number of wells required for specimens and controls was determined. An appropriate number of commercially available strips of pre-coated wells were removed from the packaging and placed in a 96-well frame provided by the manufacturer. Care was taken so as to not scratch the bottom surface of the wells as scratches could interfere with the reading of the test results.
One hundred microliters of Reagent D (capture buffer composed of 8 pmol biotinylated oligonucleotide in 4X SSC, 0.2% PVP) were added to the amplification blank, positive amplification control and the patient specimens, and incubated at 41°C (± 1°C) water bath for 30 minutes. The controls were set up in a 96-well plate format in the following order:
Row A, Column 1 - positive amplification control Row B, Column 1 - positive hybridization control Row C, Column 1 - empty well
Row D, Column 1 - amplification blank
One hundred microliters of the positive amplification control solution were pipetted into the center of one well (row A, column 1) and the remainder was stored at -70°C for up to 2 weeks. The container of positive hybridization control solution was vortexed for approximately 10 seconds, and 2 drops
(where one drop was approximately 50 ± 10 μl) of the positive hybridization solution was added to the center of the well indicated positive hybridization control. One hundred microliters of the solution from the microcentrifuge tube labelled "amplification blank" was pipetted into the center of the well indicated as the amplification blank. One hundred microliters of each amplified specimen was pipetted into the center of separate wells. The remaining specimen solutions were stored at -70 °C for up to two weeks for retesting if necessary.
The microwell plate was covered with a plate lid and incubated at room temperature for 20 minutes. After the incubation, the plate lid was removed and each well was washed 3 times with 300 μl wash solution (2X SSC, 0.05% Tween-20, 0.01% thimerosal) using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 2 (detection reagent composed of 1 pmol peroxidase labelled detector oligonucleotide in 30% glycerol, 5X SSC, 0.1% PVP, and 1% BSA) was promptly added to the center of each well except the empty substrate control well. The microwell plate was covered with the lid and incubated at room temperature for 30 minutes. Again the plate lid was removed and each well washed 3 times with 300 μl wash solution using a microwell plate washer or wash bottle. Following the last wash, the plate was inverted and tapped on a paper towel to remove excess wash solution. Two drops of Reagent 3 (peroxidase substrate containing 0.04% (w/v) 3,3',5,5' tetramethylbenzidine
(TMB) and 0.02% hydrogen peroxide; solution manufactured by Kirkegaard & Perry, Gaithersburg, MD) was promptly added to the center of each well including the empty substrate control well. The microwell plate was covered with the plate lid and incubated at room temperature for 15 ± 2 minutes. After the peroxidase substrate addition, the plate reader (e.g. Baxter PRIMA or
Tecom) was turned on and the absorbance set to 450 nm. After the substrate incubation was completed, 2 drops of stop solution (1 M phosphoric acid) was added into each well. The wells were then read in the plate reader within 15 minutes of stopping the reaction. Representative results of a CML EPA using clinical specimens are shown below. Specimens were processed according to the clinical trial protocol described above. One-half of the specimen was reserved for analysis by slot blot (see Example 8, Figures 12 and 13) and the remainder was used in the EPA. Examples of the EPA results of both bcr-abl translocations are as follows:
Specimen number bcr3-abl2 OD
92-548 Yes 3.9
280310 Yes 3.7 92-555 Yes 4.0
Specimen number bcr2-abl2 OD
15-089-018 Yes 3.4 222979 Yes 3.6
92-173 Yes 4.0 213053 Yes 4.0 250887 Yes 3.9
The results demonstrate that both translocations from clinical samples can be amplified and detected using the CML EPA described above. Figure 14 summarizes additional clinical test results in which the results using the CML EPA can be compared to results using other tests. The data in Figure 14 reflect a sensitivity of 91% and a specificity of 100%. Sensitivity is defined as the proportion of people who truly have a specific disease and are so identified by a test. Specificity is defined as the proportion of people who are truly free of a specific disease and are so identified by a test. As a patient relapses or enters cytogenetic remission, the relative absorbance values change. That is, when a patient enters cytogenetic remission (i.e. absence or reduction in number of cells with Ph chromosomes), the OD goes down, whereas when a patient relapses, the OD rises. These changes in the
OD correlate with what would be expected of a valid clinical assay for CML. This test can also be used prognostically to assess cytogenetic remission in patients with chronic myelogenous leukemia.
EXAMPLE 8
Slot Blot Procedure Amplification reactions were performed as in Example 7 above. An aliquot of each amplification reaction, representing 1/10 of the total volume, was denatured in 90 μl of 7.4% formaldehyde and 10X SSC at 65°C in a water bath for 10 minutes. Aliquots were immediately ice-chilled and loaded onto a Schleicher and Schuell Minifold apparatus. Nucleic acids in the aliquots were immobilized on the nitrocellulose by baking at 80 °C under vacuum conditions. The filters were pre-wetted with hybridization solution (6X SSC, 10X Denhardts, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS) and then hybridized at 68°C for 45 minutes with a 32p. labeled oligonucleotide probe that was complementary to the junction sequence of bcr2-abl2 (BB302) or bcr3-abl2 (BB303).
After hybridization, the filters were washed three times at room temperature for 5 minutes each using 1 ml buffer /cm2 filter, where the buffer was 2X SSC, 0. 1% SDS. The filters were exposed to X-ray film at -70°C with one intensifying screen. The results of the 3SR amplification using clinical specimens are shown in Figure 12 and 13. As shown in the slot blot, nucleic acid specific for the bcr-abl translocation and isolated from clinical specimens can be amplified and detected. Further, it can be distinguished whether the cells contain the bcr2-abl2 or the bcr3-abl2 translocation.
The foregoing detailed description has been provided for a better understanding of the invention only and no unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.
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(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:13: GGAGCTGCAG ATGCTGACCA AC 22
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(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:14: GCTGAAGGGC TTTTGAACTC TGCTTA 26
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(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:15 TAAGCAGAGT TCAAAAGCCC TTCAGC 26
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(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:18: AATTTAATAC GACTCACTAT AGGGAAGACT GTCCACAGCA TTCCG 45
(2) INFORMATION FOR SEQ ID NO:19
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) A AUUTTHHOORRSS:: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL:
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: * (H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:19: CACTGGCCAC AAAATCATAC AGTGC 25
(2) INFORMATION FOR SEQ ID NO:20
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:20: AATTTAATAC GACTCACTAT AGGGATGCAG ATGCTGACCA ACTCGTG 47
(2) INFORMATION FOR SEQ ID NO:21
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(X) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:21: AATTTAATAC GACTCACTAT AGGGAATGCT GACCAACTCG TGTGTGA 47
(2) INFORMATION FOR SEQ ID NO:22
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: ** [[mmaaxxiimi um of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:22: AATTTAATAC GACTCACTAT AGGGAAGATG CTGACCAACT CGTGTGT 47
(2) INFORMATION FOR SEQ ID NO:23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: * (C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:23: AATTTAATAC GACTCACTAT AGGGAGCTGA CCAACTCGTG TGTGAAA 47
(2) INFORMATION FOR SEQ ID NO:24
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations] (I) FILING DATE: *
(J) PUBLICATION DATE: * (K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:24: CGAAAAGGTT GGGGTCATTT 20
(2) INFORMATION FOR SEQ ID NO:25
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:25: TGCAACGAAA AGGTTGGGGT 20 (2) INFORMATION FOR SEQ ID NO:26
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES:
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:26 TACAGTGCAA CGAAAAGGTT 20
(2) INFORMATION FOR SEQ ID NO:27
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: 1inear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maxii
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:27: AATCATACAG TGCAACGAAA AG 22
(2) INFORMATION FOR SEQ ID NO:28
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: * (D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:28: CAAAATCATA CAGTGCAACG AA 22
(2) INFORMATION FOR SEQ ID NO:29
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations] (I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:29: CCACAAAATC ATACAGTGCA AC 22
(2) INFORMATION FOR SEQ ID NO:30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:30: AATTTAATAC GACTCACTAT AGGGATCAGG GTGCACAGCC GCAACGGC 48 (2) INFORMATION FOR SEQ ID NO:31
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:31: TCACTGGGTC CAG 13
(2) INFORMATION FOR SEQ ID NO:32
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maxi:
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:32: TCCTTGGAGT TCCAACGAG 19
(2) INFORMATION FOR SEQ ID NO:33
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: * (D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:33: AGCGAGAAGG TTTTCCT 17
(2) INFORMATION FOR SEQ ID NO:34
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL : *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations] (I) FILING DATE: * - 53 ■
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:34: CGAGCGGCTT CACTCAG 17
(2) INFORMATION FOR SEQ ID NO:35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maxim
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:35: GATGCTACTG GCCGCTGAAG GGC 23 (2) INFORMATION FOR SEQ ID NO:36
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL : *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:36: TTTTTTGATG CTACTGGCCG CTGAAGGGC 29
(2) INFORMATION FOR SEQ ID NO:37
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maxi
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:37: CTTCCTTATT GATGGTCA 18
(2) INFORMATION FOR SEQ ID NO:38
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: * (E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:38: GGTCAGCGGA ATGCTGTGGA 20
(2) INFORMATION FOR SEQ ID NO:39
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION : [list all publications in which this sequence appears]
(A) AUTHORS: * [ma_
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER [for patent-type citations]
(I) FILING DATE: * (J) PUBLICATION DATE: * (K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:39: TCATCATCTT CCTTATTGAT 20
(2) INFORMATION FOR SEQ ID NO:40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:40: ACATTCAGAA ACCCATAGAG 20
(2) INFORMATION FOR SEQ ID NO:41 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:41: GTGGACAGTC TGGAGTTT 18
(2) INFORMATION FOR SEQ ID NO:42
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: ** [[mmaaxxiimrrum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:42: CCTTATTGAT GGTCAGCG 18
(2) INFORMATION FOR SEQ ID NO:43
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1097
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: * - 60 -
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:43:
AGAACTTCCT GTCCAGCATC AATGAGGAGA TCACACCCCG ACGGCAGTCC ATGACGGTGA 60
AGAAGGGAGA GCACCGGCAG CTGCTGAAGG ACAGCTTCAT GGTGGAGCTG GTGGAGGGGG 120
CCCGCAAGCT GCGCCACGTC TTCCTGTTCA CCGAGCTGCT TCTCTGCACC AAGCTCAAGA 180
AGCAGAGCGG AGGCAAAACG CAGCAGTATG ACTGCAAATG GTACATTCCG CTCACGGATC 240
TCAGCTTCCA GATGGTGGAT GAACTGGAGG CAGTGCCCAA CATCCCCCTG GTGCCCGATG 300
AGGAGCTGGA CGCTTTGAAG ATCAAGATCT CCCAGATCAA GAGTGACATC CAGAGAGAGA 360
AGAGGGCGAA CAAGGGCAGC AAGGCTACGG AGAGGCTGAA GAAGAAGCTG TCGGAGCAGG 420
AGTCACTGCT GCTGCTTATG TCTCCCAGCA TGGCCTTCAG GGTGCACAGC CGCAACGGCA 480
AGAGTTACAC GTTCCTGATC TCCTCTGACT ATGAGCGTGC AGAGTGGAGG GAGAACATCC 540
GGGAGCAGCA GAAGAAGTGT TTCAGAAGCT TCTCCCTGAC ATCCGTGGAG CTGCAGATGC 600
TGACCAACTC GTGTGTGAAA CTCCAGACTG TCCACAGCAT TCCGCTGACC ATCAATAAGG 660
AAGATGATGA GTCTCCGGGG CTCTATGGGT TTCTGAATGT CATCGTCCAC TCAGCCACTG 720
GATTTAAGCA GAGTTCAAAA GCCCTTCAGC GGCCAGTAGC ATCTGACTTT GAGCCTCAGG 780
GTCTGAGTGA AGCCGCTCGT TGGAACTCCA AGGAAAACCT TCTCGCTGGA CCCAGTGAAA 840
ATGACCCCAA CCTTTTCGTT GCACTGTATG ATTTTGTGGC CAGTGGAGAT AACACTCTAA 900
GCATAACTAA AGGTGAAAAG CTCCGGGTCT TAGGCTATAA TCACAATGGG GAATGGTGTG 960
AAGCCCAAAC CAAAAATGGC CAAGGCTGGG TCCCAAGCAA CTACATCACG CCAGTCAACA 1020
GTCTGGAGAA ACACTCCTGG TACCATGGGC CTGTGTCCCG CAATGCCGCT GAGTATCTGC 1080 TGAGCAGCGG GATCAAT 1097 (2) INFORMATION FOR SEQ ID NO:44
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION: [list all publications in which this sequence appears]
(A) AUTHORS: * [maximum of first 10 authors]
(B) TITLE: *
(C) JOURNAL: *
(D) VOLUME: *
(E) ISSUE: *
(F) PAGES: *
(G) DATE: *
(H) DOCUMENT NUMBER: * [for patent-type citations]
(I) FILING DATE: *
(J) PUBLICATION DATE: *
(K) RELEVANT RESIDUES: *
(xi) A SEQUENCE DESCRIPTION: SEQ ID NO:44: GCTGAAGGGC TTCTTCCTTA TTGATG 26

Claims

What is claimed is: 1. A method for detecting or monitoring chronic myelogenous leukemia (CML) cells in a human patient, said CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA, comprising: a) providing RNA from hematopoietic cells of said patient; b) forming a reaction product by subjecting said RNA to RNA- based nucleic acid amplification using a pair of primers capable of causing amplification of both said bcr2-abl2 and bcr3-abl2 translocation regions; c) forming a capture mixture by contacting said reaction product with a capture agent comprising a capture oligonucleotide and a binding ligand, said capture oligonucleotide being specific for said bcr2-abl2 and bcr3- abl2 translocation regions; d) contacting said capture mixture with a solid phase having a receptor specific for said binding ligand coupled thereto; e) washing said solid phase; f) contacting said washed solid phase with a detector agent comprising a detector oligonucleotide specific for said bcr2-abl2 or bcr3-abl2 translocation regions and a detector label; and g) correlating the amount of said labeled detector oligonucleotide bound to said solid phase with the presence or quantity of said CML cells in said patient.
2. The method of claim 1, wherein said pair of primers comprises a sense- RNA oligonucleotide containing a polymerase binding site and an antisense- RNA oligonucleotide lacking a polymerase binding site.
3. The method of claim 2, wherein said pair of primers is selected from the group consisting of BB323/BB329, BB326/BB313 and BB325/BB329.
4. The method of claim 3, wherein said pair of primers is BB325/BB329.
5. The method of claim 1, wherein said binding ligand is biotin and said receptor specific for said binding ligand is selected from the group consisting of avidin and streptavidin.
6. The method of claim 1, wherein said capture oligonucleotide is selected from the group consisting of CAP6, CAP7, CAP8, CAP10, CAP14 and CAP14T.
7. The method of claim 6, wherein said capture oligonucleotide is CAP14T.
8. The method of claim 1, wherein said RNA-based nucleic acid amplification of step (b) uses self sustained sequence replication.
9. The method of claim 1, wherein step (c) is carried out at about 42°C.
10. The method of claim 1, wherein step (f) is carried out at room temperature.
11. The method of claim 1, wherein said detector oligonucleotide is selected from the group consisting of DET1, DET350, DET351, DET352, DET1- 46 and DET1-3.
12. The method of claim 11, wherein said detector oligonucleotide is DET1.
13. The method of claim 1, wherein said detector label is selected from the group consisting of horseradish peroxidase, fluorescein, and alkaline phosphatase.
14. The method of claim 13, wherein said detector label is horseradish peroxidase.
15. The method of claim 1, wherein said solid phase is selected from the group consisting of microwell plates, plastic beads, glass beads and magnetic particles.
16. A method for detecting or monitoring CML cells in a human patient, said CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA, comprising: a) providing RNA from hematopoietic cells of said patient; b) forming a reaction product by subjecting said RNA to RNA- based nucleic acid amplification using a pair of primers capable of causing amplification of both said bcr2-abl2 and bcr3-abl2 translocation regions; c) incubating said reaction product, a capture agent comprising a capture oligonucleotide specific for said bcr2-abl2 and bcr3-abl2 translocation regions and a binding ligand, and a detector agent comprising an oligonucleotide specific for said bcr2-abl2 or bcr3-abl2 translocation regions and a detector label to form a target complex; d) applying said target complex to a solid phase having a receptor specific for said binding ligand coupled thereto; e) washing said solid phase; and f) correlating the amount of said detector agent bound to said solid phase with the presence or quantity of said CML cells in said patient.
17. The method of claim 16, wherein said pair of primers comprises a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site.
18. The method of claim 17, wherein said pair of primers is selected from the group consisting of BB323/BB329, BB326/BB313 and BB325/BB329.
19. The method of claim 18, wherein said pair of primers is BB325/BB329.
20. The method of claim 16, wherein said binding ligand is biotin and said receptor specific for said binding ligand is selected from the group consisting of avidin and streptavidin.
21. The method of claim 16, wherein said capture oligonucleotide is selected from the group consisting of CAP6, CAP7, CAP8, CAP10, CAP14 and CAP14T.
22. The method of claim 21, wherein said capture oligonucleotide is CAP14T.
23. The method of claim 16, wherein said RNA-based nucleic acid amplification of step (b) is self sustained sequence replication.
24. The method of claim 16, wherem said detector oligonucleotide is selected from the group consisting of DET1, DET350, DET351, DET352, DET1- 46 and DET1-3.
25. The method of claim 24, wherein said detector oligonucleotide is DET1.
26. The method of claim 16, wherein said detector label is selected from the group consisting of horseradish peroxidase, fluorescein, and alkaline phosphatase.
27. The method of claim 26, wherein said detector label is horseradish peroxidase.
28. The method of claim 16, wherein said solid phase is selected from the group consisting of microwell plates, plastic beads and glass beads.
29. A method for detecting or monitoring CML cells in a human patient, said CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA, comprising: a) providing RNA from hematopoietic cells of said patient; b) forming a reaction product by subjecting said RNA to RNA- based nucleic acid amplification using a pair of primers capable of causing amplification of both said bcr2-abl2 and bcr3-abl2 translocation regions; c) providing a solid phase having a capture agent coupled thereto, said capture agent being specific for said bcr2-abl2 and bcr3-abl2 translocation regions; d) adding said reaction product to said coated solid phase forming a solid phase complex; e) washing said solid phase complex; f) contacting said washed solid phase complex with a detector agent comprising an oligonucleotide specific for said bcr2-abl2 or bcr3-abl2 translocation regions and a detector label; and g) correlating the amount of said detector agent bound to said solid phase with the presence or quantity of said CML cells in said patient.
30. The method of claim 29, wherein said pair of primers comprises a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site.
31. The method of claim 30, wherein said pair of primers is selected from the group consisting of BB323/BB329, BB326/BB313 and BB325/BB329.
32. The method of claim 31, wherein said pair of primers is BB325/BB329.
33. The method of claim 29, wherein said binding ligand is biotin and said receptor specific for said binding ligand is selected from the group consisting of avidin and streptavidin.
34. The method of claim 29, wherein said capture oligonucleotide is selected from the group consisting of CAP6, CAP7, CAP8, CAP10, CAP14 and CAP14T.
35. The method of claim 34, wherein said capture oligonucleotide is CAP14T.
36. The method of claim 29, wherein said RNA-based nucleic acid amplification of step (b) is self sustained sequence replication.
37. The method of claim 29, wherein said detector oligonucleotide is selected from the group consisting of DET1, DET350, DET351, DET352, DET1- 46 and DET1-3.
38. The method of claim 37, wherein said detector oligonucleotide is DET1.
39. The method of claim 29, wherein said detector label is selected from the group consisting of horseradish peroxidase, fluorescein, and alkaline phosphatase.
40. The method of claim 39, wherein said detector label is horseradish peroxidase.
41. The method of claim 29, wherein said solid phase is selected from the group consisting of microwell plates, plastic beads, glass beads and magnetic particles.
42. A method for detecting or monitoring CML cells in a human patient, said CML cells carrying either a bcr2-abl2 translocation region or a bcr3-abl2 translocation region in the chromosomal DNA, comprising: a) providing RNA from hematopoietic cells of said patient; b) forming a reaction product by subjecting said RNA to RNA- based nucleic acid amplification using a pair of primers capable of causing amplification of both said bcr2-abl2 and bcr3-abl2 translocation regions; c) incubating said reaction product with a labeled detector oligonucleotide specific for said bcr2-abl2 or bcr3-abl2 translocation regions to form a reaction product/detector complex; d) providing a solid phase having a capture agent coupled thereto, said capture agent being specific for said bcr2-abl2 and bcr3-abl2 translocation regions; e) adding said reaction product /detector complex to said coated solid phase forming a solid phase complex; f) washing said solid phase complex; and g) correlating the amount of said labeled detector oligonucleotide bound to said solid phase with the presence or quantity of said CML cells in said patient.
43. The method of claim 42, wherein said pair of primers comprises a sense-RNA oligonucleotide containing a polymerase binding site and an antisense-RNA oligonucleotide lacking a polymerase binding site.
44. The method of claim 43, wherein said pair of primers is selected from the group consisting of BB323/BB329, BB326/BB313 and BB325/BB329.
45. The method of claim 44, wherein said pair of primers is BB325/BB329.
46. The method of claim 42, wherein said binding ligand is biotin and said receptor specific for said binding ligand is selected from the group consisting of avidin and streptavidin.
47. The method of claim 42, wherein said capture oligonucleotide is selected from the group consisting of CAP6, CAP7, CAP8, CAP10, CAP14 and CAP14T.
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