Method of determining the presence and quantifying the number of di- and trinucleotide repeats

• US 5,650,277 A
• Filed: 10/04/1994
• Issued: 07/22/1997
• Est. Priority Date: 07/02/1992
• Status: Expired due to Fees

• Current Assignee: Diagenetics Limited
• Original Assignee: Diagenetics Limited
• Inventors: Eyal, Nurit, Navot, Nir
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/317,431
• Time in Patent Office: 1,022 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.52, 536/24-33, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

A method aimed at the quantification of di- and trinucleotide repeat which includes (a) treating a sample containing the nucleic acids of interest to obtain unpaired nucleotide bases spanning the position of the repeats and flanking regions, if the nucleic acids are not already single stranded; (b) contacting the unpaired nucleotide bases with an oligonucleotide primer capable of hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest preferably 3'"'"' of the trinucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest; (c) providing means to ensure that the examined nucleic acid and the oligonucleotide primer are confined to a reaction chamber at all further steps; (d) contacting the duplex with a primer extension unit which is capable of base pairing with the first nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (e) eliminating non-incorporated primer extension units; (f) contacting the template primer duplex with a primer extension unit which is capable of base pairing with the second nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (g) eliminating non-incorporated primer extension units; (h) contacting the template primer hybrid with a primer extension unit which is capable of base pairing with the third nucleotide base in the core sequence of the repeats; a detection moiety containing, primer extension unit which is capable of base pairing with a nucleotide base 5'"'"' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats; and a template dependent extension enzyme; (i) eliminating non-incorporated primer extension units; (j) detecting for the presence of detection moiety containing primer extension unit; (k) steps (d) to (j) are repeated until detecting said detection moiety; (l) the number of repeats as stated under (k) enables the determination of the number of trinucleotide repeats, therefore enabling determination of the exact repetition number.

Claims

1. A method of quantifying the number of trinucleotide repeats in a nucleic acid of interest, comprising the steps of:

(a) if the nucleic acid of interest is double stranded, treating the nucleic acid of interest to obtain unpaired nucleotide bases spanning the trinucleotide repeats and their flanking regions, or, if the nucleic acid of interest is single stranded, directly employing step (b);

(b) contacting the unpaired nucleotide bases spanning the number of trinucleotide repeats and their flanking regions with an oligonucleotide primer for hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest;

(c) providing means for confining the nucleic acid of interest and the oligonucleotide primer to a reaction chamber at all further steps (d) through (k); and

further comprising the cycled steps of;

(d) contacting the template primer duplex with a first primer extension unit for base pairing with one of the nucleotide bases, in the core sequence of the trinucleotide repeats, and with a template dependent extension enzyme;

(e) eliminating non-incorporated units of said first primer extension units;

(f) contacting the template primer duplex, which primer is now extended by one unit as described in step (d), with a second primer extension unit for base pairing with a second nucleotide base, in the core sequence of the repeat, said second nucleotide base being located adjacent to and immediately 5'"'"' of the nucleotide base employed under step (d), and with a template dependent extension enzyme;

(g) eliminating non-incorporated units of said second primer extension units;

(h) contacting the template primer duplex, which primer is now elongated by one further additional unit as described in step (f), with;

(i) a third primer extension unit for base pairing with a third nucleotide base, in the core sequence of the repeat, said third nucleotide base being located adjacent to and immediately 5'"'"' of the nucleotide base under step (f);

(ii) a detection moiety which is conjugated with a fourth primer extension unit for base pairing with a nucleotide base 5'"'"' of the repeats, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats, said detection moiety which is conjugated with said fourth primer extension unit may be present in selected cycles of this stage; and

(iii) a template dependent extension enzyme;

(i) eliminating non-incorporated units of said third and fourth primer extension units;

(j) if step (h) included said detection moiety which is conjugated with said fourth primer extension unit, detecting the presence of said detection moiety; and

if no detection is obtained,

(k) repeating steps (d) to (j) until said detection moiety is detected, said detection of said detection moiety being indicative of the number of trinucleotide repeats included in the nucleic acid of interest.

2. A method as in claim 1, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

3. A method as in claim 1 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

4. A method as in claim 1, wherein the oligonucleotide primer is substantially complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

5. A method as in claim 1, wherein the oligonucleotide primer is fully complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

6. A method as in claim 1, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple-immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

7. A method as in claim 1, wherein the extension moiety of said first, second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'"'"'-OH containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

8. A method as in claim 1, wherein the extension moiety of said first, second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'"'"'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

9. A method as in claim 1, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

10. A method as in claim 1, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

11. A method as in claim 1, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

12. A method as in claim 1, wherein steps (d) and (f) and steps (e) and (g) are combined under two successive single steps, in the first combined single step said template-primer duplex is contacted with the first and second primer extension units for base pairing with two adjacent nucleotide bases in the core sequence of the repeats, and a template dependent extension enzyme, while in the second combined single step said non-incorporated first and second primer extension units are eliminated.

13. A method as in claim 12, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

14. A method as in claim 12 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

15. A method as in claim 12, wherein the oligonucleotide primer is substantially complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

16. A method as in claim 12, wherein the oligonucleotide primer is fully complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

17. A method as in claim 12, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple-immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

18. A method as in claim 12, wherein the extension moiety of said first second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'"'"'-OH containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

19. A method as in claim 12, wherein the extension moiety of said first second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'"'"'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

20. A method as in claim 12, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

21. A method as in claim 12, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

22. A method as in claim 12, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

23. A method as in claim 1, wherein steps (f) and (h) and steps (g) and (i) are combined under two single steps, in the first combined single step said template-primer duplex, now said primer extended by one unit, is contacted with:

(i) said second and third primer extension units for base pairing with two adjacent nucleotide bases in the core sequence of the repeats;

(ii) a detection moiety which is conjugated with said fourth primer extension unit, said detection moiety which is conjugated with said fourth primer extension unit may be present in selected said cycles of this stage; and

(iii) a template dependent extension enzyme;

while in the second combined single step, said non-incorporated second third and fourth primer extension units are eliminated.

24. A method as in claim 23, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

25. A method as in claim 23 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

26. A method as in claim 12, wherein the oligonucleotide primer is fully complementary to said stretch of nucleotide based present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

27. A method as in claim 23, wherein the oligonucleotide primer is fully complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

28. A method as in claim 23, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

29. A method as in claim 23, wherein the extension moiety of said first, second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'"'"'-OH containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

30. A method as in claim 23, wherein the extension moiety of said first, second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'"'"'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

31. A method as in claim 23, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

32. A method as in claim 23, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

33. A method as in claim 23, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

35. A method aimed at simultaneous quantification of the number of nucleotide repeats having a core sequence which includes two or three nucleotides, in two alleles, each of said alleles containing any number of said repeats wherein steps of incorporating primer extension units complementary to the core sequence of the repeats are performed as derailed under claim 1, 12, 23 or 34 until a detection of said detection moiety incorporated onto said primer annealed to a nucleic acid associated with the allele containing a lower number of repeats is made, and steps of incorporating primer extension units complementary to the core sequence of the repeats are continued until a further detection of a detection moiety incorporated onto said primer annealed to the nucleic acid associated with the allele containing a higher number of repeats is made.

36. A method as in claim 35, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

37. A method as in claim 35 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

38. A method as in claim 35, wherein the oligonucleotide primer is substantially complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified.

39. A method as in claim 35, wherein the oligonucleotide primer is fully complementary to said stretch of nucleotide bases present in the nucleic acid of interest partially 3'"'"' of the nucleotide repeats to be quantified.

40. A method as in claim 35, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

41. A method as in claim 35, wherein the extension moiety of said first, second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'"'"'-OH containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

42. A method as in claim 35, wherein the extension moiety of said first, second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'"'"'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

43. A method as in claim 35, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

44. A method as in claim 35, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

45. A method as in claim 35, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

34. A method of quantifying the number of dinucleotide repeats in a nucleic acid of interest, comprising the steps of:

(a) if the nucleic acid of interest is double stranded, treating the nucleic acid of interest to obtain unpaired nucleotide bases spanning the dinucleotide repeats and their flanking regions, or, if the nucleic acid of interest is single stranded, directly employing step (b);

(b) contacting the unpaired nucleotide bases spanning the number of dinucleotide repeats and their flanking regions with an oligonucleotide primer for hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3'"'"' of the nucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest;

(c) providing means for confining the nucleic acid of interest and the oligonucleotide primer to a reaction chamber at all further steps (d)-(i); and

further comprising the cycled steps of;

(d) contacting the template primer duplex with a first primer extension unit for base pairing with one of the nucleotide bases, in the core sequence of the dinucleotide repeats, and with a template dependent extension enzyme;

(e) eliminating non-incorporated units of said first primer extension units;

(f) contacting the template primer duplex, which primer is now extended by one unit as described in step (d), with;

(i) a second primer extension unit for base pairing with a second nucleotide base, in the core sequence of the repeat, said second nucleotide base being located adjacent to and immediately 5'"'"' of the nucleotide base under step (d);

(ii) a detection moiety which is conjugated with a third primer extension unit for base pairing with a nucleotide base 5'"'"' of the repeats, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the dinucleotide repeats, said detection moiety which is conjugated with said third primer extension unit may be present in selected cycles of this stage; and

(iii) a template dependent extension enzyme;

(g) eliminating non-incorporated units of said second and third primer extension units;

(h) if step (f) included said detection moiety which is conjugated with said third primer extension unit, detecting the presence of said detection moiety; and

if no detection is obtained,

(i) repeating steps (d) to (h) until said detection moiety is detected, said detection of said detection moiety being indicative of the number of dinucleotide repeats included in the nucleic acid of interest.