Source: https://patents.google.com/patent/US6110678
Timestamp: 2018-02-24 20:24:13
Document Index: 716199626

Matched Legal Cases: ['Application No. 60', '§ 119', '§ 1', '§ 9', '§ 1', '§ 9', '§1', '§7', '§9', '§10', '§11']

US6110678A - Two-step hybridization and capture of a polynucleotide - Google Patents
US6110678A
US6110678A US09070998 US7099898A US6110678A US 6110678 A US6110678 A US 6110678A US 09070998 US09070998 US 09070998 US 7099898 A US7099898 A US 7099898A US 6110678 A US6110678 A US 6110678A
US09070998
Jay H. Shaw
This application claims priority to U.S. Provisional Application No. 60/045,430, filed May 2, 1997, under 35 U.S.C. § 119(e).
According to one aspect of the invention, there is disclosed a method of capturing a target polynucleotide present in a sample. The method includes the steps of incubating a mixture comprising a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe:target hybridization complex that includes the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe:capture probe hybridization complex that includes the immobilized probe and the capture probe; and then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex that includes the immobilized probe, the capture probe and the target polynucleotide. In one embodiment, the first incubating step uses a temperature below a Tm of the capture probe:target hybridization complex and above a Tm of the immobilization probe:capture probe hybridization complex, and the second incubating step uses a temperature below a Tm of the immobilization probe:capture probe hybridization complex. Preferably, the second incubating step is achieved by lowering the temperature of the first hybridization condition by at least about 10° C., or by at least about 20° C. In one preferred embodiment the first incubating step uses a temperature of about 60° C. and the second incubating step uses a temperature of about 40° C. or lower. Alternatively, the first incubating step may use a solution having a chemical stringency that favors formation of the capture probe:target hybridization complex and disfavors formation of the immobilization probe:capture probe hybridization complex, and the second incubating step lowers the chemical stringency of the solution, thereby favoring formation of the immobilization probe:capture probe hybridization complex. One embodiment of the method also includes the step of purifying the immobilized probe:capture probe:target hybridization complex. Another embodiment includes the step of detecting the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex. Preferably, the detecting step comprises hybridizing a labeled probe to the target polynucleotide in the purified immobilized probe:capture probe:target hybridization complex. In another embodiment, the detecting step further includes removing the labeled probe that has not hybridized to the target polynucleotide. One embodiment of the method also includes the step of detecting the target polynucleotide in the immobilized probe:capture probe:target hybridization complex, preferably by hybridizing a labeled probe to the target polynucleotide. This embodiment may also include the step of removing the labeled probe that has not hybridized to the target polynucleotide. Another embodiment of the method includes the step of amplifying the target polynucleotide to produce an amplified nucleic acid. Preferably, the amplifying step is accomplished by transcription-associated amplification. This embodiment may further include the step of detecting the amplified nucleic acid. Preferably, the detecting step includes hybridizing a labeled probe to the amplified nucleic acid that has a sequence complementary to the target polynucleotide sequence, and may also include removing the labeled probe that has not hybridized to the amplified nucleic acid. In one embodiment of the method the immobilized probe includes a capture probe-binding region of at least five nucleotide base recognition groups in length, and the capture probe includes an immobilized probe-binding region of at least five nucleotide base recognition groups in length, provided that the capture probe-binding region is complementary to the immobilized probe-binding region. Preferably, the capture probe-binding region of the immobilized probe includes (a) a first backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and (b) at least ten nucleotide base recognition groups joined to the first backbone, wherein each nucleotide base recognition group is capable of hydrogen bonding with adenine, guanine, cytosine, thymine, uracil or inosine; and the immobilized probe-binding region of the capture probe includes (a) a second backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and (b) at least ten nucleotide base recognition groups joined to the second backbone, which are capable of hydrogen bonding to the nucleotide base recognition groups joined to the first backbone. In a preferred embodiment, the capture probe-binding region of the immobilized probe consists of a repetitious base sequence of at least 10 nucleotide base recognition groups, and the immobilized probe-binding region of the capture probe consists of a repetitious base sequence comprising at least 25 nucleotide base recognition groups, of which at least 10 nucleotide base recognition groups are complementary to the capture probe-binding region. In one embodiment, the capture probe-binding region includes a sequence of about fourteen contiguous A or T, and the immobilized probe-binding region includes a sequence of 30 bases complementary thereto. In the method, the capture probe and the immobilized probe may each be made up of deoxynucleotides, ribonucleotides, 2'-methoxy substituted nucleotides, 2'-halo substituted nucleotide components, or combinations thereof.
An immobilized probe provides means for joining a capture probe to an immobilized support. The immobilized probe is a base sequence recognition molecule joined to a solid support which facilitates separation of bound target polynucleotide from unbound material. Any known solid support may be used, such as matrices and particles free in solution. For example, solid supports may be nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene and, preferably, magnetically attractable particles. Particularly preferred supports are magnetic spheres that are monodisperse (i.e., uniform in size ± about 5%), thereby providing consistent results, which is particularly advantageous for use in an automated assay.
Base sequence recognition molecules contain sequence information that permits hybridization to sufficiently complementary nucleic acids in appropriate reaction conditions. By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to a base sequence recognition molecule (e.g., another contiguous nucleic acid base sequence) by hydrogen bonding between complementary bases. The complementary base sequence may be complementary at each position in the base sequence recognition molecule using standard base pairing (e.g., G:C, A:T or A:U pairing). Alternatively, the complementary base sequence may contain one or more residues that are not complementary using standard hydrogen bonding (including abasic "nucleotides"), but the entire complementary base sequence is capable of specifically hybridizing with the base sequence recognition molecule in appropriate hybridization condition. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition, or can be determined empirically by using routine testing (e.g., See Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57). These base sequence recognition molecules may contain additional groups not providing sequence information. The additional groups, if present, do not prevent hybridization of a base sequence recognition molecule to a sufficiently complementary nucleic acid in the reaction conditions.
Tm can be predicted using standard calculations and measured using routine testing techniques well known in the art. Such methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57), and Hogan et al., U.S. Pat. No. 5,547,842.
Both strands (350 pmols each) were mixed in a 40 μL volume containing 20 μL of 2× Hybridization Buffer (200 mM lithium succinate (pH 5.1-5.2), 17% lithium lauryl sulfate (LLS), 3 mM ethylenediaminetetraacetic acid (EDTA), and 3 mM ethylene glycol N,N,N',N'-tetraacetic acid (EGTA)) and heated at 55° C. for 30 minutes. Then, each complex was diluted to 300 μl with 1× Hybridization Buffer (i.e., half the concentration of 2× Hybridization Buffer).
TABLE 1______________________________________  Hybrid T.sub.m______________________________________  dA.sub.40 :dT.sub.40         70.3° C.  dA.sub.30 :dT.sub.40         68.4° C.  dA.sub.25 :dT.sub.40         64.5° C.  dA.sub.20 :dT.sub.40         61.5° C.  dA.sub.15 :dT.sub.40         56.0° C.  dA.sub.10 :dT.sub.40         50.4° C.______________________________________
As seen from these results, as the length of complementarity increased, the Tm of the complex also increased. The longer hybridization complexes (e.g., dA30 :dT40) tended to have relatively broad absorbance shifts because there are potentially many types of hybridization complexes in the mixture due to offset pairing (e.g., 30-mer, 29-mer, 28-mer pairing and so forth). The combinations of homopolymers that resulted in shorter hybridization complexes (e.g., dA15 :dT40 which can form up to a 15-mer complex) had a Tm below about 60° C.
This property was further examined by incubating 100 μg of immobilized poly-dT14 or dT30 probe attached to magnetic beads with 2.5 pmols of a 32 P-labeled dA30 capture probe in solution. Reactants were incubated in a hybridization buffer (3 mM EDTA, 3 mM EGTA, 17% LLS, 190 mM succinic acid, 250 mM lithium hydroxide, pH 5.1±0.1) for 30 min at either 60° C. or room temperature (about 25° C.) in five replicates for each hybridization condition. At room temperature, the average percentages of the labeled dA30 probes hybridized to the dT14 and dT30 probes were 90% and 88%, respectively, whereas at 60° C. the average percentages of the labeled dA30 probes hybridized to the dT14 and dT30 probes were 61% and 1%, respectively.
TABLE 2__________________________________________________________________________             Immobilized Probe                        Immobilized ProbePoly - T of     Poly - A of             Added Before Second                        Present During BothImmobilized Probe     Capture Probe             Hybridization                        Hybridizations__________________________________________________________________________dT.sub.30 dA.sub.30             .sup. 54,895.sup.1                        31,608     dA.sub.15             44,558     30,290dT.sub.14 dA.sub.30             .sup. 65,313.sup.2                        63,240     dA.sub.15             43,285     37,479__________________________________________________________________________ .sup.1 Represents capture of 62% of the labeled target. .sup.2 Represents capture of 67% of the labeled target.
The basic protocol included the following steps. A lysate of the sample was prepared by adding a clinical sample (e.g., 500 μl of sediment from sputum, bronchoalveolar lavage or bronchial washings) to an equal volume of a lysis buffer (2.2 M LiCl, 250 mM HEPES buffer, pH 7.5, containing 4% (w/v) LLS) and organisms in the lysate were heat killed (95° C. for 15 min). If M. tuberculosis is present in the clinical sample, a target polynucleotide (e.g., rRNA sequence) derived from M. tuberculosis will be present in the lysate. An aliquot (250 μl) of the lysate was mixed with an equal volume of a solution containing the capture probe specific for the M. tuberculosis target sequence and an immobilized probe attached to a solid support. The capture probe was a 58-base oligonucleotide containing a 5' 15-base sequence complementary to a M. tuberculosis is rRNA sequence, an internal T3, and a 3'dA40 tail. The immobilized probe was a poly-dT14 sequence attached using carbodiimide chemistry (substantially as previously described by Lund, et al., Nuc. Acids Res.16:10861-10880, 1988) to a solid support of magnetic particles (0.7-1.05μ, particles, Seradyn, Indianapolis, Ind.). In this assay, 5 pmol of the capture probe and 50 μg of immobilized probe particles were used per reaction. The mixture was incubated successively at two different temperatures (60° C. for 20 min, and 25° C. for 15 min). At the first temperature, the M. tuberculosis-complementary sequence of the capture probe hybridized to the target sequence because the Tm of that hybridization complex is greater than 60° C., and at the second temperature the homopolymeric immobilized probe hybridized to the complementary homopolymeric region of the capture probe because the Tm of the dA:dT complex is less than about 50° C. Following both incubations, the magnetic beads were separated from the solution using a magnetic field substantially as described in Example 2. If M. tuberculosis was present in the sample, these magnetic beads have attached a hybridization complex consisting of immobilized probe:capture probe:target polynucleotide. If M. tuberculosis was not present in the sample, the beads have attached a hybridization complex consisting of immobilized probe and capture probe. The beads were washed twice with 1 ml of washing buffer per wash by resuspending the beads in buffer and then repeating the magnetic separation step. Washed beads were then resuspended in 75 μl of a nucleic acid amplification reagent solution for transcription associated amplification using methods substantially as described in Kacian et al., U.S. Pat. Nos. 5,399,491 and 5,554,516. The beads and 15 pmol of each primer specific for the M. tuberculosis target polynucleotide were incubated in a reaction mixture (40 mM Trizma base, pH 7.5, 17.5 mM KCl, 20 mM MgCl2, 5% polyvinylpyrrolidone (PVP), 1 mM each dNTP, 4 mM each rNTP), covered with a layer (200 μl) of inert oil to prevent evaporation, at 60° C. for 10-15 min, and then at 41.5-42° C. for 5 min. Reverse transcriptase (about 750 Units and about 2,000 Units of T7 RNA polymerase in 25 μl) was added per reaction, mixed, and the amplification of the target polynucleotide proceeded at 41.5-42° C. for 2 hr. Amplified M. tuberculosis target sequences were detected using an AE-labeled probe which was detected as chemiluminescence and expressed in relative light units (RLU) substantially as described previously (U.S. Pat. No. 5,658,737 at column 25, lines 27-46; Nelson et al., 1996, Biochem. 35:8429-8438 at 8432). For each assay, a negative control consisting of all of the same reagents but substituting an equal volume of negative sputum for sample, and a positive control consisting of all of the same reagents but including 50 μl of extracted total cellular M. tuberculosis RNA (containing about 2000 copies of rRNA) instead of sample. Duplicate tests were perfomed (RLU No.1 and No.2 in Table 3).
TABLE 3__________________________________________________________________________Sample No.    Smear Result            Culture Result                    RLU No. 1                           RLU No. 2__________________________________________________________________________1        Positive            Unknown 3.04 × 10.sup.6                           2.87 × 10.sup.62        Positive            Unknown 3.09 × 10.sup.6                           2.99 × 10.sup.63        Positive            Unknown 3.18 × 10.sup.6                           3.00 × 10.sup.64        Positive            Unknown 3.28 × 10.sup.6                           3.23 × 10.sup.65        Positive            Positive                    2.97 × 10.sup.6                           3.04 × 10.sup.66        Positive            Positive                    3.16 × 10.sup.6                           2.94 × 10.sup.67        Unknown Positive                    2.87 × 10.sup.6                           2.98 × 10.sup.68        Negative            Positive                    3.46 × 10.sup.4                           2.05 × 10.sup.69        Negative            Positive                    3.11 × 10.sup.6                           2.99 × 10.sup.610       Negative            Positive                    3.09 × 10.sup.6                           3.09 × 10.sup.611       Negative            Positive                    1.35 × 10.sup.6                           6.15 × 10.sup.512       Negative            Positive                    3.01 × 10.sup.6                           3.13 × 10.sup.613       Negative            Positive                    2.90 × 10.sup.6                           2.88 × 10.sup.614       Negative            Positive                    3.12 × 10.sup.6                           3.07 × 10.sup.615       Negative            Positive                    2.93 × 10.sup.6                           3.00 × 10.sup.6Positive Controls    Not Applicable            Not Applicable                    2.98 × 10.sup.6                           2.89 × 10.sup.6                    3.00 × 10.sup.6Negative Controls    Not Applicable            Not Applicable                    3.45 × 10.sup.3                           5.67 × 10.sup.3                    2.05 × 10.sup.3__________________________________________________________________________
The basic protocol used includes the following steps. For each assayed sample, a 20 μl aqueous solution of either 0 fg (negative control), 5 fg or 50 fg of N. gonorrhoeae target polynucleotide in 400 μl of water was mixed with 400 μl of a synthetic urine control (KOVATROL™; Hycor Biomedical, Inc.) and 200 μl of a target capture buffer (TCB; consisting of 2.2 M LiCl, 250 mM HEPES buffer, pH 7.5) containing a capture oligonucleotide (6.25 nM) having a sequence complementary to the N. gonorrhoeae target polynucleotide and a dT3 dA30 sequence. Immobilized dT14 probe attached to magnetic particles as the solid support were added to 100 μg/ml and the mixture was incubated successively at two different temperatures (60° C. for 20 min, and 25° C. for 15 min). At the first temperature, the capture probe and the target polynucleotide hybridized because the Tm of the capture probe:target polynucleotide complex is greater than 60°, and at the second temperature the immobilized probe and the poly-dA portion of the capture probe hybridized because the Tm of the dA:dT complex is less than about 52° C. Following incubation, the magnetic beads were separated from the solution using a magnetic field substantially as described in Example 2. For samples containing the N. gonorrhoeae target polynucleotide, the magnetic beads have attached the immobilized probe:capture probe:target polynucleotide complex, whereas for the negative control sample the beads have attached the immobilized probe:capture probe. Then the beads were washed twice substantially as described in Example 3 and the washed beads were resuspended in 75 μl of a nucleic acid amplification reagent and amplified substantially as described in Example 3 using a primer specific for the N. gonorrhoeae target polynucleotide. After amplification, the amplified target sequences were detected using a AE-labeled probe which was detected using a chemiluminescent assay as described in Example 3, and the signal was expressed in relative light units (RLU). For each day's assays, background RLU of the negative controls and positive controls were determined in the same manner using the same reagents. Results of the assays are shown in Table 4, including RLU results for each experimental sample, and the mean values of two positive controls and ten negative controls obtained for each day. The background (mean of two samples) values were 6.81×102, 1.18×103 and 6.61×102 RLU for days 1, 2 and 3 respectively.
TABLE 4______________________________________Sample   Target (fg)              Day 1 RLU Day 2 RLU                                Day 3 RLU______________________________________1        5         7.78 × 10.sup.5                        6.46 × 10.sup.5                                6.95 × 10.sup.52        5         7.73 × 10.sup.5                        7.08 × 10.sup.5                                7.06 × 10.sup.53        5         8.30 × 10.sup.5                        7.00 × 10.sup.5                                6.57 × 10.sup.54        5         7.69 × 10.sup.5                        7.34 × 10.sup.5                                7.60 × 10.sup.55        5         7.73 × 10.sup.5                        7.76 × 10.sup.5                                7.95 × 10.sup.56        5         7.01 × 10.sup.5                        6.47 × 10.sup.5                                7.14 × 10.sup.57        5         7.32 × 10.sup.5                        6.70 × 10.sup.5                                7.63 × 10.sup.58        5         7.84 × 10.sup.5                        7.23 × 10.sup.5                                7.06 × 10.sup.59        5         7.45 × 10.sup.5                        7.17 × 10.sup.5                                7.18 × 10.sup.510       5         7.45 × 10.sup.5                        7.11 × 10.sup.5                                7.68 × 10.sup.511       50        8.58 × 10.sup.5                        7.51 × 10.sup.5                                7.57 × 10.sup.512       50        8.20 × 10.sup.5                        4.61 × 10.sup.5                                7.77 × 10.sup.513       50        7.66 × 10.sup.5                        6.99 × 10.sup.5                                7.21 × 10.sup.514       50        7.85 × 10.sup.5                        7.97 × 10.sup.5                                7.45 × 10.sup.515       50        8.17 × 10.sup.5                        7.80 × 10.sup.5                                7.76 × 10.sup.516       50        7.98 × 10.sup.5                        7.33 × 10.sup.5                                7.42 × 10.sup.517       50        7.51 × 10.sup.5                        7.36 × 10.sup.5                                7.41 × 10.sup.518       50        7.76 × 10.sup.5                        8.01 × 10.sup.5                                7.80 × 10.sup.519       50        7.24 × 10.sup.5                        7.27 × 10.sup.5                                7.62 × 10.sup.520       50        7.74 × 10.sup.5                        7.54 × 10.sup.5                                7.70 × 10.sup.5Negatives (10)    0         201       -218    74______________________________________
Briefly, the two-step hybridization assay protocol was as follows. Each reaction tube contained 200 μl of TCB (as defined in Example 4) containing 6.25 pmols of a capture oligonucleotide having a sequence complementary to the C. trachomatis target polynucleotide and a dT3 dA30 sequence, into which was added 800 μl of prepared urine sample (400 μl urine plus 400 μl TM buffer (100 mM (NH4)2 SO4, 50 mM Hepes, pH 7.5, 8% LLS)) or 800 μl of controls (negative controls were 400 μl KOVATROL™ plus 400 μl TM buffer; positive controls were 400 μl KOVATROL™ plus 400 μl TM buffer containing 5 fg of a C. trachomatis total cellular RNA (i.e., rRNA target polynucleotide). The tubes were sealed, mixed, and incubated at 60° C. for 30 min, and then at 40° C. for 30 min. The tubes were then subjected to a magnetic field and the magnetic beads with immobilized probe were separated from the solution, and the immobilized components were washed twice substantially as described in Example 2. At 60° C., the capture probe and the C. trachomatis target polynucleotide hybridized because the Tm of the capture probe:target polynucleotide complex is greater than 60° C., and at the second temperature the immobilized poly-dT probe and the poly-A portion of the capture probe hybridized because the Tm of the dA:dT complex is less than about 52° C. The target polynucleotide was amplified using a primer specific for the C. trachomatis target sequence and reagents for transcription-associated amplification contained in a volume of 75 μl substantially as described in Example 3 except that amplification was for 1 hr. The AE-labeled probe (100 μl) specific for the C. trachomatis target sequence was added, mixed, and the mixture incubated at 60° C. for 20 min, after which RLU were detected substantially as described in Example 3.
TABLE 5__________________________________________________________________________Sample No. PCR Results        RLU   Sample No.                     PCR Results                            RLU__________________________________________________________________________1     Positive        4.42 × 10.sup.5              17     Negative                            02     Negative        1.66 × 10.sup.3              18     Positive                            3.59 × 10.sup.53     Positive        2.95 × 10.sup.5              19     Positive                            3.64 × 10.sup.54     Negative        0     20     Positive                            3.51 × 10.sup.55     Positive        1.62 × 10.sup.5              21     Positive                            3.75 × 10.sup.56     Positive        4.15 × 10.sup.5              22     Negative                            9.15 × 10.sup.37     Positive        5.76 × 10.sup.5              23     Positive                            3.21 × 10.sup.58     Negative        3.69 × 10.sup.3              24     Negative                            09     Positive        4.45 × 10.sup.5              25     Positive                            4.59 × 10.sup.510    Negative        0     26     Negative                            811    Positive        5.00 × 10.sup.5              27     Positive                            3.07 × 10.sup.512    Negative        0     28     Positive                            3.52 × 10.sup.513    Negative        0     29     Negative                            014    Negative        0     30     Negative                            015    Negative        0     Positive                     Not Applicable                            3.14 × 10.sup.5              Controls16    Negative        0     Negative                     Not Applicable                            0              Controls__________________________________________________________________________
The assays were performed substantially as described in Examples 3-6, where the samples consisted of normal urine (no bacterial contamination) that contained either: no target polynucleotide (negative controls); 5 fg of a C. trachomatis target polynucleotide; 5 fg of a N. gonorrhoeae target polynucleotide; or a mixture of 5 fg of a C. trachomatis target polynucleotide and 5 fg of a N. gonorrhoeae target polynucleotide. For each set of samples, the assay tubes further included either 0%, 1%, 5% or 10% v/v blood. Simultaneous detection of the two target sequences using chemiluminescent probes was substantially as described by Nelson et al. (1996, Biochem. 35:8429-8438 at 8432) using an ortho-F-AE labeled probe specific for the C. trachomatis target polynucleotide and a 2-Me-AE labeled probe specific for the N. gonorrhoeae target polynucleotide. The urine samples were prepared and incubated at 60° C. for 30 min, and then at 40° C. for 30 min for the successive hybridization steps substantially as described in Example 5 herein, except that the blood contaminant was included in some of the samples as described above. The immobilized probes in hybridization complexes were purified using the magnetic field and washing steps, and the purified target sequences were amplified using transcription-associated amplification procedures that included primers specific for the C. trachomatis target polynucleotide and the N. gonorrhoeae target polynucleotide as described in Examples 4 and 5 above. The detection reagent was added and simultaneous chemiluminescence was detected from the C. trachomatis-specific labeled probe the N. gonorrhoeae-specific labeled probe. For each type of sample assayed, four replicates were assayed and the RLU results (mean values for each type of sample) are presented in Table 6. In Table 6, the signal (RLU) detected for the C. trachomatis-specific labeled probe is indicated by "CT" and the signal detected for the N. gonorrhoeae-specific labeled probe is indicated by
TABLE 6__________________________________________________________________________                          C. trachomatis +  Negative          C. trachomatis                  N. gonorrhoeae                          N. gonorrhoeae  Control Target  Target  Target__________________________________________________________________________No Blood  CT: 1   CT: 2.18 × 10.sup.5                  CT: 4.90 × 10.sup.3                          CT: 2.22 × 10.sup.5  NG: 1.86 × 10.sup.2          NG: 4.05 × 10.sup.2                  NG: 9.37 × 10.sup.5                          9.37 × 10.sup.51% Blood  CT: 52  CT: 2.73 × 10.sup.5                  CT: 4.64 × 10.sup.3                          CT: 1.73 × 10.sup.5  NG: 4.41 × 10.sup.2          NG: 1.38 × 10.sup.3                  NG: 9.54 × 10.sup.5                          NG: 9.80 × 10.sup.55% Blood  CT: 27  CT: 1.91 × 10.sup.5                  CT: 0   CT: 1.65 × 10.sup.5  NG: 4.42 × 10.sup.2          NG: 1.66 × 10.sup.3                  NG: 1.06 × 10.sup.6                          NG: 9.19 × 10.sup.510% Blood  CT: 14  CT: 2.13 × 10.sup.5                  CT: 0   CT: 1.35 × 10.sup.5  NG: 1.36 × 10.sup.2          NG: 1.85 × 10.sup.3                  NG: 1.05 × 10.sup.6                          NG: 1.12 × 10.sup.6__________________________________________________________________________
1. A method of capturing a target polynucleotide present in a sample, consisting essentially of the steps of:
a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe:target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe:capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe; and
b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide.
2. The method of claim 1, wherein the first incubating step uses a temperature below a Tm of the capture probe:target hybridization complex and above a Tm of the immobilized probe:capture probe hybridization complex, and wherein the second incubating step uses a temperature below a Tm of the immobilized probe:capture probe hybridization complex.
3. The method of claim 2, wherein the second incubating step comprises lowering the temperature of the first hybridization condition by at least about 10° C.
4. The method of claim 2, wherein the second incubating step comprises lowering the temperature of the first hybridization condition by at least about 20° C.
5. The method of claim 2, wherein the first incubating step uses a temperature of about 60° C. and the second incubating step uses a temperature of about 40° C. or lower.
7. A method of purifying a target polynucleotide present in a sample, consisting essentially of the steps of:
a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe:target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe:capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe;
b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide; and
c) purifying the immobilized probe:capture probe:target hybridization complex.
8. A method of detecting a target polynucleolide present in a sample, consisting essentially of the steps of:
b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe:capture probe:target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide;
c) purifying the immobilized probe:capture probe:target hybridization complex; and
d) detecting the target polynucleotide in the immobilized probe:capture probe:target hybridization complex.
9. The method of claim 8, wherein the detecting step comprises hybridizing a labeled probe to the target polynucleotide.
the immobilized probe comprises a capture probe-binding region of at least five nucleotide base recognition groups in length, and
the capture probe comprises an immobilized probe-binding region of at least five nucleotide base recognition groups in length,
and wherein the capture probe-binding region is complementary to the immobilized probe-binding region.
12. The method of claim 11, wherein the capture probe-binding region of the immobilized probe comprises:
(a) a first backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and
(b) at least ten nucleotide base recognition groups joined to the first backbone, wherein each nucleotide base recognition group is capable of hydrogen bonding with adenine, guanine, cytosine, thymine, uracil or inosine;
and wherein the immobilized probe-binding region of the capture probe comprises:
(a) a second backbone containing at least one sugar-phosphodiester linkage, or at least one peptide nucleic acid group, at least one phosphorothioate linkage, or a combination thereof, and
(b) at least ten nucleotide base recognition groups joined to the second backbone, which are capable of hydrogen bonding to the nucleotide base recognition groups joined to the first backbone.
13. The method of claim 1, wherein the capture probe and the immobilized probe each comprise deoxynucleotide, ribonucleotide, 2'-methoxy substituted nucleotide, 2'-halo substituted nucleotide components, or combinations thereof.
US09070998 1997-05-02 1998-05-01 Two-step hybridization and capture of a polynucleotide Active US6110678A (en)
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US09070998 Active US6110678A (en) 1997-05-02 1998-05-01 Two-step hybridization and capture of a polynucleotide
US09574561 Active US6280952B1 (en) 1997-05-02 2000-05-19 Two-step hybridization and capture of a polynucleotide
US09910635 Abandoned US20020028459A1 (en) 1997-05-02 2001-07-20 Two-step hybridization and capture of a polynucleotide
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