Nucleic acid derivatives

Abstract

This invention relates to a methodology for assessing the sensitivity of an HIV-1 sample to zidovudine and to diagnostic assays for use in such assessment.

We claim:  
     
       1. A method for determining the sensitivity of an HIV- 1  sample to zidovudine, which comprises: 
       (a) isolating HIV- 1  DNA extracted from human cells or HIV- 1  RNA isolated from body fluids,  
       (b) hybridizing a delectably labeled oligonucleotide to the HIV- 1  DNA isolated is step (a), the oligonucleotide having at its 3′ end at least 15 nucleotides complementary to a region of the weld  wild type DNA sequence, its corresponding RNA, to a region of the mutant DNA sequence set forth in FIG. 1, or its corresponding RNA, wherein the oligonucleotide terminates of the 3″ end with said at least 15 nucleotides at the  2328 ,  2338 ,  2772 ,  2773 , or  2784  position,  
       (c) attempting to extend the oligonucleotide at its 3′-end,  
       (d) ascertaining the presence or absence of a detestably labeled extended oligonucleotide  
       (d) correlating the presence or absence of a delectably labeled extended oligonucleotide in step (d) with the sensitivity of the HIV- 1  sample to zidovudine.  
     
     
       2. A method for determining the sensitivity of an HIV- 1  sample to zidovudine which comprises: 
       (a) isolating nucleic acid from the sample,  
       (b) hybridizing a delectably labeled oligonucleotide to the HIV- 1  nucleic acid isolated in step (a), the oligonucleotide having at least 15 nucleotides complementary to a region of the wild type DNA sequence, its corresponding RNA, to a region of the mutant DNA sequence set forth in FIG. 1, or its corresponding RNA, wherein said oligonucleotide having at least 15 nucleotides contains at least one nucleotide at the  2328 ,  2338 ,  2772 ,  2773 , or  2784  position,  
       (c) ascertaining whether or not any of the resulting hybrids of the delectably labeled oligonucleotide and nucleic acid have complementary nucleotides at one of these positions, and  
       (d) correlating the presence or absence of a delectably labeled nucleic acid hybridization product formed instep (b) with the sensitivity of an HIV- 1  sample to zidovudine.  
     
     
       3. In the method of  claim 1  or  2 , prior to step (b), the isolated nucleic acid is amplified prior to hybridization. 
     
     
       4. A method as claimed in  claim 1  or  2  wherein the detectable label on the oligonucleotide is an enzyme, radioisotope or fluorochrome. 
     
     
       5. A method for determining the sensitivity of a patient sample containing HIV- 1  to zidovudine, comprising detecting a substitution at one or more of positions  2328 ,  2338 ,  2272 ,  2773  and/or  2784  in the reverse transcriptase gene of HIV- 1  from the patient sample relative to the wild type sequence shown in FIG. 1, wherein the presence of the one or more substitutions correlates with decreased sensitivity of the sample to zidovudine. 
     
     
       6. The method of  claim 5 , wherein the substitution is detected at position  2328 . 
     
     
       7. The method of  claim 5 , wherein the substitution is detected at position  2338 . 
     
     
       8. The method of  claim 5 , wherein the substitution is detected at position  2772 . 
     
     
       9. The method of  claim 5 , wherein the substitution is detected position  2773 . 
     
     
       10. The method of  claim 5 , wherein the substitution is detected at position  2784 . 
     
     
       11. The method of  claim 5 , further comprising: correlating the presence or absence of a substitution at the one or more positions with the sensitivity of the HIV- 1  sample to zidovudine. 
     
     
       12. A method for determining the sensitivity of a patient sample containing HIV- 1  to zidovudine, comprising: 
       
         hybridizing an oligonucleotide an HIV- 1  nucleic acid from the patient sample, wherein the oligonucleotide is complementary to an HIV- 1  reverse transcriptase sequence or its complement including one or more nucleotides at positions  2328 ,  2338 ,  2772 ,  2773 , and/or  2754 :  
       
       
         detecting hybridization between the oligonucleotide and the nucleic acid to determine a nucleotide present at one or more of positions  2328 ,  2338 ,  2773  and/or  2784 , the identity of the nucleotide indicating whether the HIV sample is sensitive to zidovudine. 
       
     
     
       13. The method of  claim 12 , wherein the oligonucleotide is immobilized, to a support. 
     
     
       14. The method of  claim 13 , wherein the HIV- 1  nucleic acid is labeled and the oligonucleotide is unlabeled. 
     
     
       15. A method of screening a sample from an HIV- 1  infected patient for zidovudine sensitivity comprising the steps of: 
       ( a )  isolating HIV- 1  nucleic acid from the sample;    
       ( b )  hybridizing an oligonucleotide to the HIV- 1  nucleic acid, the oligonucleotide comprising  15  nucleotides at least partially complimentary to either a region of the wild type HIV- 1  nucleic acid or a region of mutant HIV- 1  nucleic acid, wherein the oligonucleotide contains at least one nucleotide corresponding to position  2328 ,  2338 ,  2772 ,  2773  or  2784  of HIV- 1  nucleic acid;    
       ( c )  detective a hybridization product wherein presence of hybridization product formed by the oligonucleotide at least partly complementary to the wild type HIV- 1  nucleic acid indicates zidovudine sensitivity in the sample, and presence of hybridization product formed by the oligonucleotide at least partly complementary to the mutant HIV- 1  nuclleic acid indicates zidovudine resistance in the sample.   
     
     
       16. The method according to  claim 15 , wherein the oligonucleotide probe is detectably labeled. 
     
     
       17. The method according  claim 16 , wherein the label comprise biotin. 
     
     
       18. The method according to  claim 16 , wherein the label comprises fluorochrome. 
     
     
       19. The method according to  claim 16 , wherein the label comprises an enzyme. 
     
     
       20. The method according to  claim 15 , further comprising amplifying the HIV- 1  nucleic acid from the sample near to the hybridizing step. 
     
     
       21. The method according to  claim 20 , wherein the amplifying uses a priming oligonucleotide containing a nucleotide complementary to the HIV- 1  mutant nucleic acid at its  3 ′ end. 
     
     
       22. The method according to  claim 20 , wherein the amplifying step uses at least three priming oligonucleotides. 
     
     
       23. The method according to  claim 20 , wherein the amplifying uses detectably labeled nucleotide triphosphates. 
     
     
       24. The method according to  claim 12  or  15 , wherein the HIV- 1  nucleic acid is RNA. 
     
     
       25. A method of determining zidovudine sensitivity in a sample from an HIV- 1  infected patient comprising the steps of: 
       ( a )  detecting a mutation in a nucleic acid encoding HIV- 1  reverse transcriptase protein in the sample, the mutation comprising one or more mutations within the nucleic acid encoding amino acids  67 ,  70 ,  215  and/or  219 ; and    
       ( b )  correlating detection of the mutation with zidovudine sensitivity.   
     
     
       26. The method according to  claim 25 , wherein the mutation in the nucleic acid encodes asparagine at amino acid position  67 . 
     
     
       27. The method according to  claim 25 , wherein the mutation in the nucleic acid codes asparagine at amino acid position  70 . 
     
     
       28. The method according to  claim 25 , wherein the mutation in the nucleic acid encodes phenylalanime at amino acid position  215 . 
     
     
       29. The method according to  claim 25 , wherein the mutation in the nucleic acid encodes tyrosine at amino acid position  215 . 
     
     
       30. The method according to  claim 25 , wherein the mutation in the nucleic acid encodes glutamine at amino acid position  219 . 
     
     
       31. The method according claims  5 ,  11  or  25 , wherein the HIV- 1  reverse transcriptase nucleic acid is RNA. 
     
     
       32. An oligonucleotide probe for determining a nucleotide at one or more of position  2328 ,  2338 ,  2772 ,  2773  and/or  2784  in HIV- 1  reverse transcriptase gene, wherein the probe is complementary to an HIV- 1  reverse transcriptase sequence or its complement including at least one of the positions and the probe is provided in a form suitable for determining the nucleotide at the one or more positions. 
     
     
       33. The oligonucleotide probe of  claim 32  that hybridizes to the wild type form of the reverse transcriptase gene shown in FIG. 1 or its complement. 
     
     
       34. The oligonucleotide probe of  claim 32  that hybridizes to a mutant form of the reverse transcriptase gene shown FIG. 1 or its complement. 
     
     
       35. The oligonucleotide probe of  claim 32 , wherein the probe inclines position  2328  of the HIV- 1  reverse transcriptase sequence or its complement and hybridization of the probe to the sequence or its complement determines the nucleotide occupying position  2328 . 
     
     
       36. The oligonucleotide probe of  claim 32 , wherein the probe includes position  2338  of the HIV- 1  reverse transcriptase sequence or its complement and hybridization of the probe to the sequence or its complement determines the nucleotide occupying position  2338 . 
     
     
       37. The oligonucleotide probe of  claim 32 , wherein the probe includes position  2772  of the HIV- 1  reverse transcriptase sequence or its complement and hybridization of the probe to the sequence or its complement determines the nucleotide occupying position  2772 . 
     
     
       38. The oligonucleotide prove of  claim 32 , wherein the probe includes position  2773  of the HIV- 1  reverse transcriptase sequence or its complement and hybridization of the probe to the sequence or its complement determines the nucleotide occupying position  2773 . 
     
     
       39. The oligonucleotide probe of  claim 32 , wherein the probe includes position  2784  of the HIV- 1  reverse transcriptase sequence or its complement and hybridization of the probe to the sequence or its complement determines the nucleotide occupying position  2784 .