Method for determining nucleotide identity through extension of immobilized primer

• US 6,004,744 A
• Filed: 10/11/1991
• Issued: 12/21/1999
• Est. Priority Date: 03/05/1991
• Status: Expired due to Term

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Molecular Tool, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Anderson, Stephen, Knapp, Michael R., Goelet, Philip
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US07/775,786
• Time in Patent Office: 2,993 Days
• Field of Search: 435/6, 435/91.2, 435/5, 536/26.12, 536/23.1, 536/24.33, 536/26.1
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention concerns a reagent composition that employs at least two different terminators of a nucleic acid template-dependent primer extension reaction to determine the identity of a nucleotide base at a specific position in a nucleic acid of interest. The invention also concerns an immobilized method for determining such identification. The invention may be used to determine the presence or absence of a specific nucleotide sequence in a sample. It may also be employed in determination of genotype and in the identification of different alleles.

Claims

1. A reagent composition which comprises an aqueous carrier, a primer nucleic acid molecule, a template nucleic acid of interest hybridized thereto, and an admixture of at least two different terminators of a nucleic acid template-dependent, primer extension reaction, each of the terminators being capable of specifically terminating an extension reaction of said primer hybridized to said template nucleic acid molecule of interest in a manner strictly dependent on the identity of the unpaired nucleotide base in said template nucleic acid molecule of interest immediately adjacent to, and downstream of, the 3'"'"' end of said primer, and at least one of the terminators being labeled with a detectable marker, and wherein said reagent composition lacks dATP, dCTP, dGTP, and dTTP.

2. A reagent of claim 1, wherein the reagent comprises four different terminators.

3. A reagent of claim 2, wherein two of the terminators are labeled, each with a different detectable marker.

4. A reagent of claim 2, wherein three of the terminators are labeled, each with a different detectable marker.

5. A reagent of claim 2, wherein the four terminators are labeled, each with a different detectable marker.

6. A reagent of any of claims 1-5, wherein the terminator(s) comprise(s) a nucleotide or nucleotide analog.

7. A reagent of claim 6, wherein the terminator(s) comprise(s) dideoxynucleotides.

8. A reagent of claim 6, wherein the terminator(s) comprise(s) arabinoside triphosphates.

9. A reagent of claim 7, wherein the terminator(s) comprise(s) one or more of ddATP, ddCTP, ddGTP or ddTTP.

10. A reagent of any of claims 1-5, wherein each of the different detectable markers is an isotopically labeled moiety, a chromophore, a fluorophore, a protein moiety, or a moiety to which an isotopically labeled moiety, a chromophore, a fluorophore, or a protein moiety can be attached.

11. A reagent of claim 10, wherein each of the different detectable markers is a different fluorophore.

12. A reagent of any of claims 1-5, wherein the reagent further comprises pyrophosphatase.

13. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest which comprises:

(a) treating a sample containing the nucleic acid of interest, if such nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;

(b) contacting the sample from step (a), with an oligonucleotide primer, said primer being immobilized to a solid support wherein said primer is fully complementary to and hybridizes specifically to a stretch of nucleotide bases present in the nucleic acid of interest, immediately adjacent to the nucleotide base to be identified, under high stringency hybridization conditions, so as to form a duplex between the immobilized primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately downstream of the 3'"'"' end of the primer in said duplex;

(c) contacting the duplex from step (b) in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent on the identity of the unpaired nucleotide base in the template nucleic acid of interest immediately downstream of the 3'"'"' end of the immobilized primer;

wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker, wherein if more than one terminator is labeled, a different label is used to label each such labeled terminator;

wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with the nucleotide base to be identified and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3'"'"' end of the primer to thereby extend said 3'"'"' end of said primer by one terminator;

(d) determining the presence and identity of the nucleotide base at the specific position in the nucleic acid of interest by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3'"'"' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

17. A method of typing a sample containing nucleic acids which comprises identifying the nucleotide base or bases present at each of one or more specific positions, each such nucleotide base being identified using the method of claim 13 or 15, and each such specific position being determined using a different primer.

18. A method of claim 17, wherein the identity of each nucleotide base or bases at each position is determined individually or wherein the identities of the nucleotide bases at different positions are determined simultaneously.

20. A method for identifying different alleles in a sample containing nucleic acids which comprises identifying the nucleotide base or bases present at each of one or more specific positions, each such nucleotide base being identified by the method of claim 13 or 16.

21. A method for determining the genotype of an organism at one or more particular genetic loci which comprises:

(a) obtaining from the organism a sample containing genomic DNA; and

(b) identifying the nucleotide base or bases present at each of one or more specific positions in nucleic acids of interest, each such base or bases being identified using the method of claim 13 or 15, and thereby identifying different alleles and thereby, in turn, determining the genotype of the organism at one or more particular genetic loci.

22. A method of claim 13 or 15, wherein the conditions for the occurrence of the template-dependent, primer extension reaction in step (c) are created, in part, by the presence of a suitable template-dependent enzyme.

23. A method of claim 22, wherein the template-dependent enzyme is E. coli DNA polymerase I or the "Klenow fragment" thereof, T4 DNA polymerase, T7 DNA polymerase ("Sequenase"), T. aquaticus DNA polymerase, a retroviral reverse transcriptase, or combinations thereof.

24. A method of claim 13 or 15, wherein the nucleic acid of interest is a deoxyribonucleic acid, a ribonucleic acid, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

25. A method of claim 13 or 15, wherein the primer is an oligodeoxyribonucleotide, an oligoribonucleotide, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

26. A method of claim 13 or 15, wherein the template is a deoxyribonucleic acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a copolymer of deoxyribonucleotides and ribonucleotides, and the template-dependent enzyme is a DNA polymerase.

27. A method of claim 13 or 15, wherein the template is a ribonucleic acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a copolymer of deoxyribonucleotides and ribonucleotides, and the template-dependent enzyme is a reverse transcriptase.

28. A method of claim 13 or 15, wherein the template is a deoxyribonucleic acid, the primer is an oligoribonucleotide, and the enzyme is an RNA polymerase.

29. A method of claim 13 or 15, wherein the template is a ribonucleic acid, the primer is an oligoribonucleotide, and the template-dependent enzyme is an RNA replicase.

30. A method of claim 13 or 15, wherein, prior to the primer extension reaction in step (c), the template has been capped at its 3'"'"' end by the addition of a terminator to the 3'"'"' end of the template, said terminator being capable of terminating a template-dependent, primer extension reaction.

31. A method of claim 30, wherein the terminator is a dideoxynucleotide.

32. A method of claim 13 or 15, wherein the nucleic acid of interest has been synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically.

33. A method of claim 13 or 15, wherein the oligonucleotide primer has been synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically.

34. A method of claim 13 or 15, wherein the oligonucleotide primer comprises one or more moieties that permit affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest.

35. A method of claim 34, wherein the oligonucleotide primer comprises biotin which permits affinity separation of the primer from the unincorporated reagent and/or nucleic acid of interest via binding of the biotin to streptavidin which is attached to a solid support.

36. A method of claim 13 or 15, wherein the sequence of the oligonucleotide primer comprises a DNA sequence that permits affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest via base pairing to a complementary sequence present in a nucleic acid attached to a solid support.

37. A method of claim 13 or 15, wherein the nucleic acid of interest comprises one or more moieties that permit affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer.

38. A method of claim 37, wherein the nucleic acid of interest comprises biotin which permits affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer via binding of the biotin to streptavidin which is attached to a solid support.

39. A method of claim 13 or 15, wherein the sequence of the nucleic acid of interest comprises a DNA sequence that permits affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer via base pairing to a complementary sequence present in a nucleic acid attached to a solid support.

40. A method of claim 13 or 15, wherein the oligonucleotide primer is labeled with a detectable marker.

41. A method of claim 40, wherein the oligonucleotide primer is labeled with a detectable marker that is different from any detectable marker present in the reagent or attached to the nucleic acid of interest.

42. A method of claim 13 or 15, wherein the nucleic acid of interest is labeled with a detectable marker.

43. A method of claim 42, wherein the nucleic acid of interest is labeled with a detectable marker that is different from any detectable marker present in the reagent or attached to the primer.

44. A method of claim 13 or 15, wherein the nucleic acid of interest comprises non-natural nucleotide analogs.

45. A method of claim 44, wherein the non-natural nucleotide analogs comprise deoxyinosine or 7-deaza-2'"'"'-deoxyguanosine.

46. A method of claim 13 or 15, wherein the nucleic acid of interest has been synthesized by the polymerase chain reaction.

47. A method of claim 13 or 15, wherein the sample comprises genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

48. A method of claim 13 or 15, wherein the sample comprises extragenomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

49. A method of claim 13 or 15, wherein the primer is separated from the nucleic acid of interest after the primer extension reaction in step (c) by using appropriate denaturing conditions.

50. A method of claim 49, wherein the denaturing conditions comprise heat, alkali, formamide, urea, glyoxal, enzymes, and combinations thereof.

51. A method of claim 50, wherein the denaturing conditions comprise treatment with 0.2 N NaOH.

52. A method of claim 47, wherein the organism is a plant, microorganism, virus, or bird.

53. A method of claim 47, wherein the organism is a vertebrate or invertebrate.

54. A method of claim 47, wherein the organism is a mammal.

55. A method of claim 54, wherein the mammal is a human being.

56. A method of claim 54, wherein the mammal is a horse, dog, cow, cat, pig, or sheep.

14. A method of determining the presence or absence of a desired polynucleotide having a particular nucleotide sequence in a sample of nucleic acids which comprises:

(a) treating a sample of nucleic acids, if such sample of nucleic acids contains double-stranded nucleic acids, so as to obtain single-stranded nucleic acids, or directly employing step (b) if the sample of nucleic acids contains only single-stranded nucleic acids;

(b) contacting the sample from step (a), with an oligonucleotide primer, said primer being immobilized to a solid support;

wherein said primer is fully complementary to and hybridizes specifically to a stretch of nucleotide bases of the particular nucleotide sequence, if the desired polynucleotide having said particular nucleotide sequence is present, said contacting being under high stringency hybridization conditions, so as to form a duplex between the immobilized primer and the particular nucleotide sequence;

(c) contacting the duplex, if any, from step (b) in the absence of dATP, dCTP, dGTP or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent on the identity of the unpaired nucleotide base in the template immediately downstream of the 3'"'"' end of the immobilized primer;

wherein one of said terminators is complementary to said unpaired nucleotide base and wherein at least one of said terminators is labeled with a detectable marker, wherein if more than one terminator is labeled, a different label is used to label each such labeled terminator;

wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with said unpaired nucleotide base immediately downstream of the 3'"'"' end of said immobilized hybridized primer and occurrence of a template-dependent, primer extension reaction sufficient to incorporate said complementary terminator onto the 3'"'"' end of the immobilized primer to thereby extend said 3'"'"' end of said primer by one terminator; and

(d) determining the presence of the target nucleic acid molecule by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3'"'"' end of the extended primer from step (c) and wherein said detection is conducted in the absence of non-terminator nucleotides.

19. A method of typing a sample containing nucleic acids which comprises determining the presence or absence of one or more particular nucleotide sequences, the presence or absence of each such nucleotide sequence being determined by the method of claim 14 or 16.

15. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest which comprises:

(a) (1) incubating a sample containing the nucleic acid of interest with at least two oligonucleotide primers, and a polymerase, said primers being sufficient to mediate a polymerase chain reaction amplification of said nucleic acid of interest, wherein said incubation is conducted under conditions sufficient to permit said amplification to occur;

(2) treating a sample containing said amplified nucleic acid of interest, if such nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;

(b) contacting the sample from step (a2), under hybridizing conditions, with an oligonucleotide primer, said primer being immobilized to a solid support wherein said primer is fully complementary to and hybridizes specifically to a stretch of nucleotide bases present in the nucleic acid of interest immediately adjacent to the nucleotide base to be identified, so as to form a duplex between the immobilized primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately downstream of the 3'"'"' end of the primer in said duplex;

(c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template nucleic acid of interest immediately downstream of the 3'"'"' end of the immobilized primer;

wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker, wherein if more than one terminator is labeled, a different label is used to label each such labeled terminator;

wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with the nucleotide base immediately downstream of the 3'"'"' end of said hybridized primer and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3'"'"' end of the primer to thereby extend said 3'"'"' end of said primer by one terminator;

(d) determining the presence and identity of the nucleotide base at the specific position in the nucleic acid of interest by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3'"'"' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

16. A method of determining the presence or absence of a desired polynucleotide having a particular nucleotide sequence in a sample of nucleic acids which comprises:

(a) (1) incubating a sample of nucleic acids with at least two oligonucleotide primers, and a polymerase, said primers being sufficient to mediate a polymerase chain reaction amplification of said nucleic acid of interest, if said nucleic acid of interest is present in said sample, wherein said incubation is conducted under conditions sufficient to permit said amplification to occur;

(2) treating a sample containing said amplified nucleic acid of interest, if such nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;

(b) contacting the sample from step (a2), under hybridizing conditions, with an oligonucleotide primer, said primer being immobilized to a solid support;

wherein said primer is fully complementary to and hybridizes specifically to a stretch of nucleotide bases of the particular nucleotide sequence, if the desired polynucleotide having said particular nucleotide sequence is present, so as to form a duplex between the immobilized primer and the particular nucleotide sequence;

(c) contacting the duplex, if any, from step (b), in the absence of dATP, dCTP, dGTP or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template immediately downstream of the 3'"'"' end of the immobilized primer;

wherein one of said terminators is complementary to said unpaired nucleotide base and wherein at least one of said terminators is labeled with a detectable marker, wherein if more than one terminator is labeled, a different label is used to label each such labeled terminator;

wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with said unpaired nucleotide base immediately downstream of the 3'"'"' end of said immobilized hybridized primer and occurrence of a template-dependent, primer extension reaction sufficient to incorporate said complementary terminator onto the 3'"'"' end of the immobilized primer to thereby extend said 3'"'"' end of said primer by one terminator; and

(d) determining the presence of the target nucleic acid molecule by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3'"'"' end of the extended primer from step (c) and wherein said detection is conducted in the absence of non-terminator nucleotides.