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.

1. A method for determining the sensitivity of an HIV- 1  samples to zidovudine, which comprises:
 (a) isolating HIV- 1  DNA extracted from human cells or HIV- 1  RNA isolated from body fluids,    (b) hybridizing a detectably labeled oligonucleotide to the HIV- 1  DNA isolated in step (a), the oligonucleotide having at its 3′ end at least 15 nucleotides complementary to a region of the weld 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 detectably labeled extended oligonucleotide,    (d) correlating the presence or absence of a detectably labeled extended oligonucleotide in step (d) with the sensitivity of the HIV- 1  samples 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 detectably 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 detectably labeled oligonucleotide and nucleic acid have complementary nucleotides at one of these positions, and    (d) correlating the presence of absence of a detectably 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 ,  2772 ,  2773  and/or 2784 in the reverse transcriptase gene or HIV-1 from the patient sample relative to the wildtype sequence shown in FIG. 1, wherein the presence of 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 at position 2773. 
     
     
       10. The method of  claim 5 , wherein the substitution is detected at position 2784. 
     
     
       11. The method of  claim 5 , further comprising:
 assessing the sensitivity of the HIV sample to zidovudine from the presence or absence of a substitution at one or more of the nucleotides.    
     
     
       12. A method for determining the sensitivity of a patient sample containing HIV-1 to zidovudine, comprising:
 hybridizing an oligonucleotide to a HIV-1 nucleic acid from the patient sample, wherein the oligonucleotide is complementary to a HIV-1 reverse transcriptase sequence or its complement including one or more nucleotides as positions  2328 ,  2338 ,  2772 ,  2773 , and/or 2784;    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 labelled and the oligonucleotide is unlabelled. 
     
     
       15. An oligonucleotide probe for determining a nucleotide at one or more of positions  2328 ,  2338 ,  2772 ,  2773  and/or 2784 in a HIV-1 reverse transcriptase gene, wherein the probe is complementary to a HIV-1 reverse transcriptase sequence or its complement including at least one of the positions. 
     
     
       16. The oligonucleotide probe of  claim 15  that hybridizes to the wildtype form of the reverse transcriptase gene shown in FIG. 1 or its complement. 
     
     
       17. The oligonucldotide probe of  claim 15  that hybridizes to a mutant form of the reverse transcriptase gene shown in FIG. 1 or its complement. 
     
     
       18. The oligonucleotide probe of  claim 15 , wherein the probe includes 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 . 
     
     
       19. The oligonucleotide probe of  claim 15 , 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 . 
     
     
       20. The oligonucleotide probe of  claim 15 , 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. 
     
     
       21. The oligonucleotide probe of  claim 15 , 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. 
     
     
       22. The oligonucleotide probe of  claim 15 , 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. 
     
     
       23. A method of screening a HIV patient being or to be treated with a drug, comprising:
 obtaining a sample from the patient;    detecting one or more substitutions in a reverse transcriptase gene of a HIV genome from the patient sample; and correlating the one or more substitutions with decreased sensitivity to the drug.    
     
     
       24. The method of  claim 23 , further comprising:
 diagnosing decreased sensitivity to the drug in a second patient infected with HIV having a reverse transcriptase gene with at least one of the substitutions.    
     
     
       25. A method for determining the sensitivity of a patient sample containing HIV-   1  to zidovudine, comprising:    ( a )  isolating a nucleic acid sample from cells of the patient;      ( b )  amplifying a reverse - transcriptase sequence from the nucleic acid sample;      ( c )  performing dideoxy sequencing of the reverse - transcriptase sequence; and      ( d )  detecting a substitution at one or more of positions  2328 ,  2338 ,  2772 ,  2773  and  2784  in the reverse transcriptase sequence of HIV -   1  from the patient sample relative to the wild type sequence shown in FIG.  1, wherein the presence of one or more substitutions correlates with decreased sensitivity of the sample to zidovudine.   
     
     
       26. The method of  claim 25 , wherein step ( b )  is performed by amplifying HIV -   1  RNA by RT - PCR.   
     
     
       27. The method of  claim 25 , wherein step ( b )  is performed by amplifying HIV -   1  DNA by PCR.   
     
     
       28. The method of  claim 25 , wherein the amplifying is performed through the use of nested primers. 
     
     
       29. The method of  claim 25 , wherein the patient has been treated with zidovudine prior to performing step ( a ). 
     
     
       30. The method of  claim 25 , wherein the patient has not been treated with zidovudine prior to performing step ( a ). 
     
     
       31. The method of  claim 25 , wherein the substitution is detected at position  2328 . 
     
     
       32. The method of  claim 25 , wherein the substitution is detected at position  2338 . 
     
     
       33. The method of  claim 25 , wherein the substitution is detected at position  2772 . 
     
     
       34. The method of  claim 25 , wherein the substitution is detected at position  2773 . 
     
     
       35. The method of  claim 25 , wherein the substitution is detected at position  2784 . 
     
     
       36. A method for determining the sensitivity of a patient sample containing HIV-   1  to zidovudine, comprising:    ( a )  hybridizing an immobilized oligonucleotide that is at least  15  nucleotides long to 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 ; and      ( b )  detecting hybridization between the oligonucleotide and the nucleic acid to determine a nucleotide present at one or more of positions  2328 ,  2338 ,  2772 ,  2773  and/or  2784 , the identity of the nucleotide indicating whether the HIV sample is sensitive to zidovudine.     
     
     
       37. The method of  claim 36 , wherein the HIV-   1  nucleic acid is labeled and the oligonucleotide is unlabeled.   
     
     
       38. The method of  claim 36 , wherein the oligonucleotide includes position  2328  of the HIV-   1  reverse transcriptase sequence or its complement and hybridization of the oligonucleotide to the sequence or its complement determines the nucleotide occupying position  2328 .   
     
     
       39. The method of  claim 36 , wherein the oligonucleotide includes position  2338  of the HIV-   1  reverse transcriptase sequence or its complement and hybridization of the oligonucleotide to the sequence or its complement determines the nucleotide occupying position  2338 .   
     
     
       40. The method of  claim 36 , wherein the oligonucleotide includes position  2772  of the HIV-   1  reverse transcriptase sequence or its complement and hybridization of the oligonucleotide to the sequence or its complement determines the nucleotide occupying position  2772 .   
     
     
       41. The method of  claim 36 , wherein the oligonucleotide includes position  2773  of the HIV-   1  reverse transcriptase sequence or its complement and hybridization of the oligonucleotide to the sequence or its complement determines the nucleotide occupying position  2773 .   
     
     
       42. The method of  claim 36 , wherein the oligonucleotide includes position  2784  of the HIV-   1  reverse transcriptase sequence or its complement and hybridization of the oligonucleotide to the sequence or its complement determines the nucleotide occupying position  2784 .   
     
     
       43. The method of  claim 36 , wherein the nucleic acid is isolated by amplifying HIV-   1  RNA by RT - PCR.   
     
     
       44. The method of  claim 36 , wherein the nucleic acid is isolated by amplifying HIV-   1  DNA by PCR.   
     
     
       45. The method of  claim 43 , wherein the amplifying is performed through the use of nested primers. 
     
     
       46. The method of  claim 44 , wherein the amplifying is performed through the use of nested primers. 
     
     
       47. The method of  claim 36 , wherein the patient has been treated with zidovudine prior to performing step ( a ). 
     
     
       48. The method of  claim 36 , wherein the patient has not been treated with zidovudine prior to performing step ( a ).