Direct detection of expanded nucleotide repeats in the human genome

• US 5,695,933 A
• Filed: 05/28/1993
• Issued: 12/09/1997
• Est. Priority Date: 05/28/1993
• Status: Expired due to Fees

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Hudson, Thomas J., Schalling, Martin, Housman, David E.
• Primary Examiner(s): Guzo, David
• Application Number:
  US08/068,747
• Time in Patent Office: 1,656 Days
• Field of Search: 435/91.52, 435/6, 436/177, 436/514, 935/77
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Methods of detecting expanded nucleotide repeats in genomic DNA in a biological sample are disclosed and methods for the diagnosis of pathological and potentially pathological conditions in an individual based on the detection of the presence of expanded nucleotide repeats.

Claims

1. A method of detecting an expanded nucleotide repeat of undetermined length in genomic DNA in a biological sample, comprising the steps of:

(a) isolating genomic DNA contained in a biological sample, thereby producing isolated genomic DNA;

(b) combining genomic DNA from step (a) with immediately ligatable simple sequence repeat oligonucleotides consisting of a nucleic acid sequence complementary to a nucleotide repeat to be detected in the genomic DNA, thereby producing a combination of genomic DNA and immediately ligatable oligonucleotides;

(c) annealing the immediately ligatable oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed oligonucleotide complexes, thereby producing genomic DNA/annealed multimer complexes;

(e) denaturing the genomic DNA/annealed multimer complexes, thereby releasing the annealed multimers from the multimer complexes and producing unannealed multimers; and

(f) repeating steps (c) through (e) to obtain a sufficient number of unannealed multimers for detection, whereby the presence of unannealed multimers is an indication of the presence of an expanded nucleotide repeat in the genomic DNA.

2. The method of claim 1 wherein the method further comprises the steps of:

(g) separating, on the basis of size, unannealed multimers by polyacrylamide gel electrophoresis;

(h) electrotransferring the gel from step (g) to a membrane suitable for hybridization thereby producing a membrane having present thereon unannealed multimers;

(i) contacting labeled oligonucleotide probes, having nucleic acid sequences complementary to the unannealed multimers, with the membrane produced in step (h) under conditions sufficient for hybridization of the labeled oligonucleotide probes to the unannealed multimers present on the membrane, whereby labeled oligonucleotide probes and unannealed multimers hybridize, thereby producing a pattern of labeled probes hybridized to unannealed multimers; and

(j) visualizing the pattern of labeled oligonucleotide probes hybridized to the unannealed multimers present on the membrane, whereby the visualized pattern is an indication of the presence and size of unannealed multimers.

3. The method of claim 1 wherein the simple sequence repeat oligonucleotides provided in step (b) are labelled with a florescent tag.

4. The method of claim 2 wherein the oligonucleotide probes of step (i) are labeled with a radioactive label and the visualization step

(j) is by autoradiography.

5. The method of claim 2 wherein the oligonucleotide probes of step (i) are labelled with a non-radioactive label.

6. The method of claim 4 wherein the radioactive label is ³² P.

7. The method of claim 5 wherein the non-radioactive label is peroxidase, biotin or digoxigenin.

8. The method of claim 3 wherein the method further comprises the steps of:

(g) separating, on the basis of size, unannealed multimers tagged with a florescent tag by polyacrylamide gel electrophoresis;

(h) detecting the presence of florescent-tagged unannealed multimers in the gel of step (g) using a fluorescence-detecting scanner.

9. A method of detecting an expanded trinucleotide repeat of undetermined length in genomic DNA in a biological sample, comprising the steps of:

(a) isolating genomic DNA contained in a biological sample, thereby producing isolated genomic DNA;

(b) combining genomic DNA from step (a) with immediately ligatable simple sequence repeat oligonucleotides consisting of a nucleic acid sequence complementary to the trinucleotide repeat to be detected in the genomic DNA, thereby producing a combination of genomic DNA and immediately ligatable oligonucleotides;

(c) annealing the immediately ligatable oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed oligonucleotide complex, thereby producing genomic DNA/annealed multimer complexes;

(e) denaturing the genomic DNA/annealed multimer complexes, thereby releasing the annealed multimers from the multimer complexes and producing unannealed multimers;

(f) repeating steps (c) through (e) to obtain a sufficient number of unannealed multimers for detection;

(g) separating, on the basis of size, unannealed multimers by polyacrylamide gel electrophoresis;

(h) electrotransferring the gel from step (g) to a membrane suitable for hybridization, thereby producing a membrane having present thereon unannealed multimers;

(i) contacting radiolabeled oligonucleotide probes having nucleic acid sequences complementary to the unannealed multimers with the membrane produced in step (h), under conditions sufficient for hybridization of the radiolabeled oligonucleotide probes to the unannealed multimers present on the membrane, whereby radiolabeled oligonucleotide probes and unannealed multimers hybridize, thereby producing radiolabeled probes hybridized to unannealed multimers; and

(j) visualizing, by autoradiography, radiolabeled oligonucleotide probes hybridized to the unannealed multimers whereby visualized radiolabeled probes are an indication of the presence of unannealed multimers and the presence of unannealed multimers is an indication of the presence of an expanded trinucleotide repeat in the genomic DNA.

10. The method of claim 9 wherein the radioactive label is ³² P.

11. The method of claim 9 wherein the simple sequence repeat oligonucleotides provided in step (b) are one or more selected from the group consisting of:

(CGG)₁₁ ;

(TGG)₁₂ ;

(CCT)₁₃ ;

(CGT)₁₄ (SEQ ID NOS;

7-10) and (CTG)₁₇ (SEQ ID NO;

1) and the labeled oligonucleotide probes of step (i) are one or more selected from the group consisting of;

(CCG)₁₀ ;

(CCA)₁₀ ;

(AGG)₁₀ ;

(ACG)₁₀ and (CAG)₁₀ (SEQ ID NOS;

2-6).

12. A method of detecting a locus-specific expanded nucleotide repeat of undetermined length in genomic DNA in a biological sample, comprising the steps of:

(a) isolating genomic DNA contained in a biological sample, thereby producing isolated genomic DNA;

(b) combining the genomic DNA from step (a) with immediately ligatable simple sequence repeat oligonucleotides consisting of a nucleic acid sequence complementary to the nucleotide repeat to be detected in the genomic DNA, and with immediately ligatable oligonucleotides consisting of a nucleic acid sequence complementary to a nucleic acid sequence located at a specific locus on the genomic DNA, thereby producing a combination of genomic DNA, immediately ligatable simple sequence repeat oligonucleotides and immediately ligatable locus-specific oligonucleotides;

(c) annealing the immediately ligatable simple sequence repeat oligonucleotides and immediately ligatable locus-specific oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed locus-specific oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed locus-specific oligonucleotide complexes, thereby producing genomic DNA/annealed locus-specific multimer complexes;

(e) denaturing the genomic DNA/annealed locus-specific multimer complexes, thereby releasing annealed locus-specific multimers from the multimer complexes and producing unannealed locus-specific multimers;

(f) repeating steps (c) through (e) to obtain a sufficient number of unannealed locus-specific multimers for detection, whereby the presence of unannealed locus-specific multimers is an indication of the presence of an expanded nucleotide repeat located at a specific locus on the genomic DNA.

13. The method of claim 12 wherein the method further comprises the steps of:

(g) separating, on the basis of size, unannealed locus-specific multimers by polyacrylamide gel electrophoresis;

(h) electrotransferring the gel from step (g) to a membrane suitable for hybridization thereby producing a membrane having present thereon unannealed locus-specific multimers;

(i) contacting labeled oligonucleotide probes having nucleic acid sequences complementary to the locus-specific unannealed multimers with the membrane produced in step (h) under conditions sufficient for hybridization of the labeled oligonucleotide probes to the unannealed locus-specific multimers present on the membrane, whereby labeled oligonucleotide probes and unannealed multimers hybridize, thereby producing labeled probes hybridized to unannealed locus-specific multimers; and

(j) visualizing the labeled oligonucleotide probes hybridized to the unannealed locus-specific multimers present on the membrane, whereby visualized labeled probes are an indication of the presence of unannealed locus-specific multimers and the presence of unannealed locus-specific multimers is an indication of the presence of a locus-specific expanded nucleotide repeat in the genomic DNA.

14. The method of claim 12 wherein the simple sequence repeat oligonucleotides provided in step (b) are labelled with a florescent tag.

15. The method of claim 12 further comprising the steps:

(g) amplifying the unannealed locus-specific multimers using linear PCR and primers;

(h) separating, on the basis of size, amplified unannealed locus-specific multimers by polyacrylamide gel electrophoresis;

(i) electrotransferring the gel from step (h) to a membrane suitable for hybridization thereby producing a membrane having present thereon unannealed locus-specific multimers;

(j) contacting labeled oligonucleotide probes having nucleic acid sequences complementary to unannealed locus-specific multimers with the membrane produced in step (i) under conditions sufficient for hybridization of the labeled oligonucleotide probes to the unannealed locus-specific multimers present on the membrane, whereby labeled oligonucleotide probes and unannealed locus-specific multimers hybridize, thereby producing labeled probes hybridized to unannealed locus-specific multimers; and

(k) visualizing the labeled oligonucleotide probes hybridized to the unannealed locus-specific multimers membrane, whereby visualized labeled probes are an indication of the presence of unannealed locus-specific multimers and the presence of unannealed locus-specific multimers is an indication of the presence of a locus-specific expanded nucleotide repeat.

16. The method of claim 13 wherein the oligonucleotide probes of step (i) are labeled with a radioactive label and the visualization step (j) is by autoradiography.

17. The method of claim 13 wherein the oligonucleotide probes of step (i) are labelled with a non-radioactive label.

18. The method of claim 16 wherein the radioactive label is ³² P.

19. The method of claim 17 wherein the non-radioactive label is peroxidase, biotin or digoxigenin.

20. The method of claim 14 wherein the method further comprises the steps of:

(g) separating, on the basis of size, unannealed locus-specific multimers tagged with a florescent tag by polyacrylamide gel electrophoresis;

(h) detecting the presence of florescent-tagged unannealed locus-specific multimers in the gel of step (g) using a fluorescence-detecting scanner.

21. The method of claim 15 wherein the oligonucleotide probes of step (j) are labeled with a radioactive label and the visualization step (k) is by autoradiography.

22. The method of claim 15 wherein the oligonucleotide probes of step (j) are labeled with a florescent tag and the visualization step (k) is by detecting the presence of florescent-tagged probes using a fluorescence-detecting scanner.

23. The method of claim 21 wherein the radioactive label is ³² P.

24. A method of detecting an expanded nucleotide repeat of undetermined length in genomic DNA in a biological sample, comprising the steps of:

(a) isolating genomic DNA from a biological sample;

(b) combining the genomic DNA from step (a) with a mixture of different types of immediately ligatable simple sequence repeat oligonucleotides, each type of oligonucleotide consisting of a nucleic acid sequence complementary to a different type of nucleotide repeat to be detected in the genomic DNA, thereby producing a combination of genomic DNA and immediately ligatable oligonucleotides;

(c) annealing the different types of immediately ligatable simple sequence repeat oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed oligonucleotide complexes, thereby producing genomic DNA/annealed multimer complexes;

(e) denaturing the genomic DNA/multimer complexes thereby releasing annealed multimers and producing unannealed multimers;

(f) repeating steps (c) through (e) to obtain a sufficient number of unannealed multimers for detection;

(g) separating, on the basis of size, unannealed multimers by polyacrylamide gel electrophoresis;

(h) electrotransferring the gel from step (g) to a membrane suitable for hybridization thereby producing a membrane having present thereon unannealed multimers;

(I) contacting a mixture of different types of radiolabeled oligonucleotide probes having nucleic acid sequences complementary to the unannealed multimers with the membrane produced in step (h) under conditions sufficient for hybridization of the radiolabeled oligonucleotide probes to the unannealed multimers present on the membrane whereby radiolabeled oligonucleotide probes and unannealed multimers hybridize, thereby producing a pattern of radiolabeled probes hybridized to unannealed multimers; and

(j) visualizing the pattern of radiolabeled oligonucleotide probes hybridized to the unannealed multimers present on the membrane by autoradiography, whereby the visualized pattern is an indication of the presence and size of unannealed multimers and the presence and size of unannealed multimers is an indication of the presence of a type of expanded nucleotide repeat in the genomic DNA.

25. The method of claim 24 wherein the radioactive label is ³² P.

26. The method of claim 24 wherein the simple sequence repeat oligonucleotides provided in step (b) are one or more selected from the group consisting of:

(CGG)₁₁ ;

(TGG)₁₂ ;

(CCT)₁₃ ;

(CGT)₁₄ (SEQ ID NOS.;

7-10) and (CTG)₁₇ (SEQ ID NO.;

1) and the labeled oligonucleotide probes of step (i) are one or more selected from the group consisting of;

(CCG)₁₀ ;

(CCA)₁₀ ;

(AGG)₁₀ ;

(ACG)₁₀ and (CAG)₁₀ ;

(SEQ ID NOS.;

2-6).

27. A method of diagnosing a potentially pathological or pathological condition in an individual by the detection of a locus-specific expanded nucleotide repeat of undetermined length characteristic of the condition in the genomic DNA of the individual comprising the steps of:

(a) isolating genomic DNA from a biological sample;

(b) combining the genomic DNA from step (a) with immediately ligatable simple sequence repeat oligonucleotides consisting of a nucleic acid sequence complementary to the nucleotide repeat characteristic of the condition to be detected in the genomic DNA, and immediately ligatable oligonucleotides consisting of a nucleic acid sequence complementary to a nucleic acid sequence located at a specific locus on the genomic DNA, thereby producing a combination of DNA, immediately ligatable simple sequence repeat oligonucleotides and immediately ligatable locus-specific oligonucleotides;

(c) annealing the immediately ligatable simple sequence repeat oligonucleotides and the immediately ligatable locus-specific oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed locus-specific oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed locus-specific oligonucleotide complexes, thereby producing genomic DNA/annealed locus-specific multimer complexes;

(e) denaturing the genomic DNA/annealed locus-specific multimer complexes, thereby releasing annealed locus-specific multimers and producing unannealed locus-specific multimers;

(f) repeating steps c through (e) to obtain a sufficient number of unannealed locus-specific multimers for detection;

(g) amplifying the unannealed locus-specific multimers using linear PCR and primers;

(h) separating, on the basis of size, amplified unannealed locus-specific multimers by polyacrylamide gel electrophoresis;

(I) electrotransferring the gel from step (h) to a membrane suitable for hybridization thereby producing a membrane having present thereon unannealed locus-specific multimers;

(j) contacting labeled oligonucleotide probes having nucleic acid sequences complementary to the locus-specific unannealed multimers with the membrane produced in step (I) under conditions sufficient for hybridization of the labeled oligonucleotide probes to the unannealed locus-specific multimers present on the membrane, thereby providing labeled probes hybridized to unannealed locus-specific multimers; and

(k) visualizing the labeled oligonucleotide probes hybridized to the unannealed locus-specific multimers present on the membrane, whereby visualized labeled probes are an indication of the presence of unannealed locus-specific multimers and the presence of unannealed locus-specific multimers is an indication of the presence of a locus-specific expanded nucleotide repeat characteristic of a potentially pathological or pathological condition.

28. The method of claim 27 wherein the oligonucleotide probes of step (j) are labeled with a radioactive label and the visualization step (k) is by autoradiography.

29. The method of claim 28 wherein the radioactive label is ³² P.

30. A kit for the diagnosis of a potentially pathological or pathological condition in an individual by the detection of an expanded nucleotide repeat of undetermined length in the genomic DNA of the individual comprising:

(a) a container of genomic DNA consisting of a known expanded nucleotide repeat sequence;

(b) a container of simple sequence repeat oligonucleotides;

(c) a container of labeled oligonucleotide probes;

(d) a container of DNA ligase enzyme; and

(e) a container of DNA ligase buffer.

31. A kit for the diagnosis of a potentially pathological or pathological condition in an individual by the detection of an expanded nucleotide repeat of undetermined length in the genomic DNA of the individual comprising:

(a) a container of genomic DNA consisting of a known expanded nucleotide repeat sequence;

(b) a container of one or more simple sequence repeat oligonucleotides selected from the group consisting of;

(CGG)₁₁ ;

(TGG)₁₂ ;

(CCT)₁₃ ;

(CGT)₁₄ (SEQ ID NOS.;

7-10) and (CTG)₁₇ ;

(SEQ ID NO.;

1);

(c) a container of one or more labeled oligonucleotide probes selected from the group consisting of;

(CCG)₁₀ ;

(SEQ ID NOS.;

2-6);

(CCA)₁₀ ;

(AGG)₁₀ ;

(ACG)₁₀ and (CAG)₁₀ ;

(d) a container of DNA ligase enzyme; and

(e) a container of DNA ligase buffer.

32. The kit of claim 31 wherein the labeled oligonucleotide probes are labeled with ³² P.

33. The kit of claim 31 wherein the labeled oligonucleotide probes are labeled with a florescent tag.

34. A method of identifying an expanded nucleotide repeat of undetermined length in genomic DNA in a biological sample which is characteristic of a genetic condition in an individual comprising the steps of:

(a) isolating genomic DNA from a biological sample;

(b) combining the genomic DNA from step (a) with immediately ligatable simple sequence repeat oligonucleotides consisting of a nucleic acid sequence complementary to the nucleotide repeat to be detected in the genomic DNA, thereby producing a combination of genomic DNA and immediately ligatable oligonucleotides;

(c) annealing the immediately ligatable oligonucleotides to the isolated genomic DNA, thereby producing genomic DNA/annealed oligonucleotide complexes;

(d) ligating the annealed oligonucleotides to produce multimers of the annealed oligonucleotides in the genomic DNA/annealed oligonucleotide complex, thereby producing genomic DNA/annealed multimer complexes;

(e) denaturing the genomic DNA/annealed multimer complexes, thereby releasing the annealed multimers from the multimer complexes and producing unannealed multimers;

(f) repeating steps (c) through (e) to obtain a sufficient number of unannealed multimers for detection;

(g) separating, on the basis of size, unannealed multimers by polyacrylamide gel electrophoresis;

(h) electrotransferring the gel from step (g) to a membrane suitable for hybridization, thereby producing a membrane having present thereon unannealed multimers;

(I) contacting labeled oligonucleotide probes having nucleic acid sequences complementary to the unannealed multimers with the membrane produced in step (h), under conditions sufficient for hybridization of the labeled oligonucleotide probes to the unannealed multimers present on the membrane, whereby label oligonucleotide probes and unannealed multimers hybridize, thereby producing labeled probes hybridized to unannealed multimers; and

(j) visualizing labeled oligonucleotide probes hybridized to the unannealed multimers whereby visualized labeled probes are an indication of the presence unannealed multimers and the presence of unannealed multimers is an indication of the presence of an expanded nucleotide repeat in the genomic DNA which is characteristic of a genetic condition in an individual.

35. A method of detecting the presence of and determining the length of an expanded nucleotide repeat in genomic DNA, comprising the steps of:

a) providing multiple copies of immediately ligatable repeat-specific oligonucleotides consisting of a nucleic acid sequence complementary to the expanded nucleotide repeat to be detected;

b) annealing the multiple copies of immediately ligatable repeat-specific oligonucleotides to the expanded nucleotide repeat, if present in the genomic DNA, to form repeat-specific oligonucleotide/expanded nucleotide repeat complexes comprising two, or more, adjacent repeat-specific oligonucleotides annealed to the expanded nucleotide repeat;

c) ligating two or more adjacent repeat-specific oligonucleotides present in the complex with a thermostable ligase to form multimers of various lengths of the annealed repeat-specific oligonucleotides;

d) denaturing the complexes to release the multimers from the genomic DNA;

e) repeating steps b) through d) to form multimers of maximum length and in sufficient number for detection; and

f) separating the multimers of various lengths on the basis of size and detecting the multimers wherein the longest length of multimer detected is indicative of the length of the expanded nucleotide repeat present in the genomic DNA.

36. The method of claim 35 wherein the repeat-specific oligonucleotides are one or more selected from the group consisting of:

(CGG)₁₁ ;

(TGG)₁₂ ;

(CCT)₁₃ ;

(CGT)₁₄ (SEQ ID NOS.;

7-10) and (CTG)₁₇ (SEQ ID NO.;

1).

37. The method of claim 35 wherein the separation of multimers on the basis of size comprises separation by polyacrylamide gel electrophoresis and the detection of multimers comprises the following steps:

a) electrotransferring the gel to a membrane suitable for hybridization, thereby producing a membrane having present thereon multimers of various lengths;

b) providing labeled oligonucleotide probes having nucleic acid sequences complementary to the multimers to be detected;

c) contacting the labeled oligonucleotide probes with the membrane of step a) under conditions sufficient for hybridization of the labeled probes to the multimers present on the membrane, whereby labeled probes hybridize to the multimers thereby producing a pattern of labeled probed hybridized to the multimers of various lengths; and

d) visualizing the pattern of labeled probes hybridized to the multimers present on the membrane, whereby the visualized pattern is an indication of the presence and length of multimers.

38. The method of claim 35 wherein the repeat-specific oligonucleotides are labeled whir a fluorescent tag.

39. The method of claim 37 wherein the labeled oligonucleotide probes are one or more selected from the group consisting of:

(CCG)₁₀ ;

(CCA)₁₀ ;

(AGG)₁₀ ;

(ACG)₁₀ and (CAG)₁₀ (SEQ ID NOS.;

2-6).

40. The method of claim 37 wherein the labeled oligonucleotide probes are labeled with a radioactive label and the visualization step is accomplished by autoradiography.

41. The method of claim 37 wherein the labeled oligonucleotide probes are labeled with a non-radioactive label.

42. The method of claim 37 wherein the labeled oligonucleotide probes are labeled with a fluorescent tag and the visualization step is accomplished by detecting the presence of fluorescence-tagged probes using a florescence-detecting scanner.

43. The method of claim 40 wherein the radioactive label is ³² P.

44. The method of claim 41 wherein the non-radioactive label is peroxidase, biotin or digoxigenin.

45. A method of detecting the presence of an expanded trinucleotide repeat sequence of undetermined length in genomic DNA, wherein the expanded nucleotide repeat in the genomic DNA acts as a template for the annealing and ligation of repeat-specific oligonucleotides, comprising the steps of:

a) denaturing the genomic DNA to obtain single-stranded DNA;

b) annealing immediately ligatable trinucleotide repeat-specific oligonucleotides to the single-stranded DNA, said repeat-specific oligonucleotides consisting of a sequence being complementary to either strand of the expanded trinucleotide repeat to be detected, said repeat-specific oligonucleotides having unmodified ends and therefore capable of direct ligation;

c) ligating two or more repeat-specific oligonucleotides annealed in adjacent positions with a thermostable ligase to form multimer of the annealed repeat-specific oligonucleotides;

d) subjecting the annealed multimer to denaturing conditions to form single-stranded multimer of repeat-specific oligonucleotides; and

e) repeating steps b) through d) to obtain a sufficient number of single-stranded multimer for detection whereby the presence of single-stranded multimer is an indication of the presence of an expanded trinucleotide repeat in the genomic DNA.