Target and background capture methods with amplification for affinity assays

Abstract

A method of assay for target polynucleotides includes steps of isolating target polynucleotides from extraneous non-target polynucleotides, debris, and impurities and amplifying the target polynucleotide.

We claim:  
     
       1. A method for amplifying a target polynucleotide contained in a sample comprising the steps of: 
       (a) contacting the sample with a first support which binds to the target polynucleotide;  
       (b) substantially separating the support and bound target polynucleotide from the sample, thereby producing a separated target polynucleotide; and  
       (c) amplifying in vitro the separated target polynucleotide of ( b ).  
     
     
       2. The method of  claim 1  wherein the first support is retrievable. 
     
     
       3. The method of  claim 1  wherein the first support includes a probe which binds with the target polynucleotide. 
     
     
       4. The method of  claim 1  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said separated target polynucleotide with a polymerase. 
     
     
       5. The method of  claim 4  wherein the polymerase is a DNA polymerase, an RNA polymerase, or a transcriptase or Qβ replicase . 
     
     
       6. The method of  claim 4  wherein the separated target polynucleotide is a DNA polynucleotide and the polymerase is a DNA polymerase. 
     
     
       7. A method for detecting a target polynucleotide contained in a sample comprising the steps of: 
       (a) contacting the sample with a first support which binds to the target polynucleotide;  
       (b) substantially separating the first support and bound target polynucleotide from the sample, thereby producing a separated target polynucleotide;  
       (c) amplifying in vitro the separated target polynucleotide of ( b ) , thereby producing an amplified target polynucleotide ; and  
       (d) detecting the presence of the amplified target polynucleotide of ( c )  as indicative of the presence of the target polynucleotide in said sample.    
     
     
       8. The method of  claim 7  wherein the first support is retrievable. 
     
     
       9. The method of  claim 8  wherein the first support includes a probe which binds with the target polynucleotide. 
     
     
       10. The method of  claim 7  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said separated target polynucleotide with a polymerase. 
     
     
       11. The method of  claim 10  wherein the polymerase is a DNA polymerase, an RNA polymerase, or a transcriptase or Qβ replicase . 
     
     
       12. The method of  claim 11  wherein the separated target polynucleotide is a DNA polynucleotide and the polymerase is a DNA polymerase. 
     
     
       13. The method of  claim 7  wherein the amplified target polynucleotide is contacted with a label, and the presence of the target polynucleotide in the sample is indicated by detection of said label. 
     
     
       14. The method of  claim 7  wherein the amplified target polynucleotide is contacted with a labeled probe, and the presence of the target polynucleotide in the sample is indicated by detection of said labeled probe. 
     
     
       15. The method of  claim 7  wherein the amplified target polynucleotide is contacted with a second support which binds to the amplified target polynucleotide. 
     
     
       16. The method of  claim 15  wherein the amplified target polynucleotide is contacted with  second support includes a labeled probe, and the presence of the target polynucleotide in the sample is indicated by detection of said labeled probe. 
     
     
       17. The method of  claim 16  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said separated target polynucleotide with a polymerase. 
     
     
       18. The method of  claim 17  wherein the separated target polynucleotide is a DNA polynucleotide and the polymerase is a DNA polymerase. 
     
     
       19. A method for detecting a target polynucleotide contained in a sample comprising the steps of: 
       (a) contacting the sample with a first support which binds to the target polynucleotide;  
       (b) substantially separating the first support and bound target polynucleotide from the sample, thereby producing a separated target polynucleotide;  
       (c) amplifying in vitro the sample  separated polynucleotide of ( b ) with a DNA polymerase, thereby producing an amplified target polynucleotide;  
       (d) contacting the amplified target polynucleotide of ( c ) with a second support which binds to the amplified target polynucleotide and also with a labeled probe which binds to the amplified target polynucleotide; and  
       (e) detecting the presence of the amplified target polynucleotide  the labeled probe as indicative of the presence of the target polynucleotide in said sample.  
     
     
       20. A kit for detecting a target polynucleotide contained in a sample comprising: 
       (a) means for substantially separating the target polynucleotide from the sample;  
       (b) means for amplifying the target polynucleotide;  
       (c) means for binding the amplified target polynucleotide to a solid support; and  
       (d) means for labeling the amplified target polynucleotide.  
     
     
       21. The kit of  claim 20  wherein: 
       (a) the means for substantially separating the target polynucleotide from the sample include a first support;  
       (b) the means for amplifying the target polynucleotide include a polymerase;  
       (c) the means for binding that amplified target polynucleotide to a solid support include a capture probe which binds to the solid support and to the amplified target polynucleotide; and  
       (d) a detector probe for labeling the amplified target polynucleotide.  
     
     
       22. The kit of  claim 21  further comprising a capture probe which binds to the first support and to the target. 
     
     
       23. The kit of  claim 22  wherein the polymerase is a DNA polymerase and the detector probe is labeled. 
     
     
       24. A kit for amplifying a target polynucleotide contained in a sample comprising: 
       (a) means for substantially separating the target polynucleotide from the sample and  
       (b) means for amplifying the target polynucleotide.  
     
     
       25. The kit of  claim 24  wherein: 
       (a) the means for substantially separating the target polynucleotide from the sample includes a support which binds to the target polynucleotide and  
       (b) the means for amplifying the target polynucleotide includes a polymerase.  
     
     
       26. The kit of  claim 25  wherein: 
       (a) the polymerase is a DNA polymerase; and  
       (b) the means for substantially separating the target polynucleotide from the sample includes a probe which binds to the target polynucleotide and the support.  
     
     
       27. A method for amplifying a target polynucleotide contained in a sample medium comprising the steps of: 
       (a) contacting the sample medium with a reagent comprising a first nucleic acid probe which binds to the target polynucleotide to form a probe-target complex;  
       (b) contacting the sample medium with a support which binds to the first nucleic acid probe of the probe-target complex;  
       (c) substantially separating the support and bound probe-target complex from the sample medium;  
       (d) contacting the support and bound probe-target complex with a second medium;  
       (e) releasing the probe-target complex into the second medium;  
       (f) substantially separating the support from the second medium; and  
       (g) amplifying in vitro the target polynucleotide in the probe- target complex present in the second medium.    
     
     
       28. A method for detecting a target polynucleotide contained in a sample medium comprising the steps of: 
       (a) contacting the sample medium with a reagent comprising a first nucleic acid probe which binds to the target polynucleotide to form a probe-target complex;  
       (b) contacting the sample medium with a support which binds to the first nucleic acid probe of the probe-target complex;  
       (c) substantially separating the support and bound probe-target complex from the sample medium;  
       (d) contacting the support and bound probe-target complex with a second medium;  
       (e) releasing the probe-target complex into the second medium;  
       (f) substantially separating the support from the second medium;  
       (g) amplifying in vitro the target polynucleotide in the probe- target complex present in the second medium ; and  
       (h) detecting the presence of the target polynucleotide in the second medium as indicative of the presence of the target polynucleotide in said sample.  
     
     
       29. The method of detecting a target polynucleotide of  claim 28  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said target polynucleotide with a polymerase. 
     
     
       30. The method for detecting a target polynucleotide of  claim 29  wherein the polymerase is a DNA polymerase, an RNA polymerase, or a transcriptase, or Qβ replicase . 
     
     
       31. The method for detecting a target polynucleotide of  claim 30  wherein the polymerase is a DNA polymerase. 
     
     
       32. The method for amplifying a target polynucleotide of  claim 27  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said target polynucleotide with a polymerase. 
     
     
       33. The method for amplifying a target polynucleotide of  claim 32  wherein the polymerase is a DNA polymerase. 
     
     
       34. A method for amplifying a target polynucleotide contained in a sample medium comprising the steps of: 
       (a) contacting the sample medium with a support and a probe which binds to the target polynucleotide and the support;  
       (b) substantially separating the support and bound probe and target polynucleotide from the sample medium;  
       (c) contacting the support and bound probe and target polynucleotide with a second medium;  
       (d) releasing the target polynucleotide of ( c ) into the second medium;  
       (e) substantially separating the support and bound probe from the second medium; and  
       (f) amplifying in vitro the target polynucleotide present in the second medium.  
     
     
       35. The method for amplifying a target polynucleotide of  claim 34  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said target polynucleotide with a polymerase. 
     
     
       36. The method for amplifying a target polynucleotide of  claim 35  wherein the polymerase is a DNA polymerase, an RNA polymerase, or a transcriptase or Qβ replicase . 
     
     
       37. The method for amplifying a target polynucleotide of  claim 36  wherein the polymerase is a DNA polymerase. 
     
     
       38. A method for detecting a target polynucleotide contained in a sample medium comprising the steps of: 
       (a) contacting the sample medium with a support and probe which binds to the target polynucleotide and the support;  
       (b) substantially separating the support and bound probe and target polynucleotide from the sample medium;  
       (c) contacting the support and bound probe and target polynucleotide with a second medium;  
       (d) releasing the target polynucleotide of ( c ) into the second medium;  
       (e) substantially separating the support and bound probe form  from the second medium;  
       (f) amplifying in vitro the target polynucleotide present in the second medium, thereby producing an amplified target polynucleotide; and  
       (g) detecting the presence of the amplified target polynucleotide in the second medium as indicative of the presence of the target polynucleotide in said sample.  
     
     
       39. The method for detecting a target polynucleotide of  claim 38  wherein the target polynucleotide is amplified with a polymerase  said amplifying in vitro comprises amplifying said target polynucleotide with a polymerase. 
     
     
       40. The method for detecting a target polynucleotide of  claim 39  wherein the polymerase is a DNA polymerase. 
     
     
       41. The amplification method of  claim 1 , wherein said amplifying it vitro is linear or exponential. 
     
     
       42. The amplification method of  claim 41  wherein said amplifying in vitro is exponential. 
     
     
       43. The amplification method of  claim 1  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with at least one oligonucleotide primer. 
     
     
       44. The amplification method of  claim 43  wherein said amplifying in vitro is linear or exponential. 
     
     
       45. The amplification method of  claim 1  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with more than one polymerase. 
     
     
       46. The detection method of  claim 7  wherein said amplifying in vitro is linear or exponential. 
     
     
       47. The detection method of  claim 46  wherein said amplifying in vitro is exponential. 
     
     
       48. The detection method of  claim 7  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with at least one oligonucleotide primer. 
     
     
       49. The detection method of  claim 48  wherein said amplifying in vitro is linear or exponential. 
     
     
       50. The detection method of  claim 7  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with more than one polymerase. 
     
     
       51. The method of  claim 1  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide non- specifically.   
     
     
       52. The method of  claim 1  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide specifically. 
     
     
       53. The method of  claim 7  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide non- specifically.   
     
     
       54. The method of  claim 7  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide specifically. 
     
     
       55. The method of  claim 9  wherein the probe first binds with the target polynucleotide by hybridizing to a specific sequence in the target polynucleotide, and then binds to the first support. 
     
     
       56. The method of  claim 55  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide non- specifically.   
     
     
       57. The method of  claim 55  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide specifically. 
     
     
       58. The method of  claim 57  wherein the sample is a clinical sample. 
     
     
       59. The method of  claim 58  wherein the probe comprises a nucleotide sequence specific to a complementary nucleotide sequence in the target polynucleotide and a homopolymeric tail sequence. 
     
     
       60. The method of  claim 59  wherein the support comprises a homopolymeric tail complementary to the homopolymeric tail of the probe. 
     
     
       61. The method of  claim 1  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with specially tailored primers. 
     
     
       62. The method of  claim 7  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with specially tailored primers. 
     
     
       63. The method of  claim 55  wherein said amplifying in vitro comprises amplifying said separated target polynucleotide with specially tailored primers. 
     
     
       64. The method of  claim 63  wherein the sample is a clinical sample.