CHEMICALLY MODIFIED OLIGONUCLEOTIDE PRIMERS FOR NUCLEIC ACID AMPLIFICATION

• US 20070281308A1
• Filed: 05/17/2007
• Published: 12/06/2007
• Est. Priority Date: 06/01/2006
• Status: Active Grant

• Current Assignee: TriLink BioTechnologies Incorporated
• Original Assignee: TriLink BioTechnologies Incorporated
• Inventors: Zon, Gerald, Lebedev, Alexandre
• Granted Patent: US 8,361,753 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

The present invention provides methods and compositions for nucleic acid amplification. These methods involve the use of oligonucleotide primers in temperature dependent nucleic acid amplification reactions. In certain aspects, the methods are accomplished by use of certain modified oligonucleotide primers which provide utility in nucleic acid amplification. In preferred embodiments, the oligonucleotide primers are modified with particular chemical groups such as esters.

Claims

1. A method of amplifying nucleic acids, said method comprising:

amplifying nucleic acid using a modified oligonucleotide primer,

wherein said modified oligonucleotide primer comprises a modification group at one or more internucleotide linkages;

wherein said modification group can be non-reversibly thermally dissociated; and

wherein said modified oligonucleotide primer has structure I as follows

wherein;

Nuc is a nucleoside within the primer sequence;

U and Z are independently O, S, Se, NR⁹, or CR⁹R¹⁰;

R⁹ and R¹⁰ are each independently hydrogen or optionally substituted straight or branched

hydrocarbyl having from 1-20 carbon atoms, wherein each may independently include at least one substituent selected from halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido or a detectable label;

Y is O, S or Se;

W is O, S, S(O), S(O)₂, Se, C(O), C(S), C(O)NH, NH or NR⁹; and

Q is a modification group comprising one or more thermally cleavable groups.

2. The method of claim 1, wherein said modification group, Q, comprises one or more modification groups selected from the group consisting of

-L-X—

R¹ 

 

(Formula I)

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms;

X is O, S, S(O), S(O)₂, C(O), C(S) or C(O)NH; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20

carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy and heteroaryl;

-L-S(O)_k—

R¹ 

 

(Formula Ib)

wherein;

k is an integer from 0-2;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon

atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

Each R¹ is independently hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20

carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;

-L-R² 

 

(Formula II)

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R² is hydrogen, cyano, or an optionally substituted carbocycle, heterocycle, aryl or heteroaryl having between

5-10 atoms;

-L^a-A-L^b-B 

 

(Formula III)

wherein

L^a and L^b are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;

A is O, S, S(O), S(O)₂, Se, CR³R⁴, NR³, C(O), C(S) or CNR³;

B is C(O)R³, C(S)R³, C(O)NR³R⁴, OR³ or SR³; and

R³ and R⁴ are each independently hydrogen or straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl; and

-L^a-D-L^b-E-L^c-F 

 

(Formula IV)

wherein;

L^a, L^b and L^c are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;

D is O, S, S(O), S(O)₂, CR⁵R⁶, and NR⁵;

E is O, S, S(O), S(O)₂, CR⁵R⁶, and NR⁶;

F is hydrogen, C(O)R⁷, C(S)R⁷, C(O)NR⁷R⁸, OR⁷ and SR⁷;

R⁵ and R⁶ can each independently be hydrogen, aryl, alkyl, halo, oxo, hydroxyl,

alkoxy, aryloxy or amino, or R⁵ and R⁶ can cooperate to form a mono or bicyclic ring consisting 5-10 atoms and including D, R⁵, R⁶, E and L^b, provided that when R⁵ and R⁶ cooperate to form a ring, n is from 0-2; and

R⁷ and R⁸ are each independently selected from the group consisting of aryl, alkyl, halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido, optionally substituted cycloalkyl, optionally substituted hetercycloalkyl, optionally substituted aryl, optionally substituted aryloxy, and optionally substituted heteroaryl.

3. The method of claim 1, wherein said modification group, Q, comprises:

-L-X—

R¹ 

 

(Formula I)

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms;

X is O, S, S(O), S(O)₂, C(O), C(S) or C(O)NH; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20

carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

4. The method of claim 1, wherein said modification group, Q, comprises:

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon

atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

5. The method of claim 1, wherein said modification group, Q, comprises:

-L-S(O)_k—

R¹ 

 

(Formula Ib)

wherein;

k is an integer from 0-2;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

6. The method of claim 1, wherein said modification group, Q, comprises:

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

Each R¹ is independently hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

7. The method of claim 1, wherein said modification group, Q, comprises:

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R¹ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon

atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

8. The method of claim 1, wherein said modification group, Q, comprises:

-L-R² 

 

(Formula II)

wherein;

L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and

R² is hydrogen, cyano, or an optionally substituted carbocycle, heterocycle, aryl or heteroaryl having between

5-10 atoms.

9. The method of claim 1, wherein said modification group, Q, comprises:

-L^a-A-L^b-B 

 

(Formula III)

wherein;

L^a and L^b are each independently a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;

A is O, S, S(O), S(O)₂, Se, CR³R⁴, NR³, C(O), C(S) or CNR³;

B is C(O)R³, C(S)R³, C(O)NR³R⁴, OR³ or SR³; and

R³ and R⁴ are each independently hydrogen or straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of hydrogen, aryl, alkyl, halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.

10. The method of claim 1, wherein said modification group, Q, comprises:

-L^a-D-L^b-E-L^c-F 

 

(Formula IV)

wherein;

L^a, L^b and L^c are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms ;

D is O, S, S (O), S(O)₂, CR⁵R⁶, and NR⁵;

E is O, S, S(O), S(O)₂, CR⁵R⁶, and NR⁶;

F is hydrogen, C(O)R⁷, C(S)R⁷, C(O)NR⁷R⁸, OR⁷ and SR⁷;

R⁵ and R⁶ can each independently be hydrogen, alkyl, aryl, halo, oxo, hydroxyl,

alkoxy, aryloxy or amino, or R⁵ and R⁶ can cooperate to form a mono or bicyclic ring consisting 5-10 atoms and including D, R⁵, R⁶, E and L^b, provided that when R⁵ and R⁶ cooperate to form a ring, n is from 0-2; and

R⁷and R⁸ are each independently selected from the group consisting of hydrogen,

alkyl, aryl, halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido, optionally substituted cycloalkyl, optionally substituted hetercycloalkyl, optionally substituted aryl, optionally substituted aryloxy, and optionally substituted heteroaryl.

11. The method of claim 1, wherein said modification group, Q, comprises one or more chemical moieties selected from the group consisting of 4-oxo-1-hexyl, 4-oxo-1-pentyl, 5-oxo-1-hexyl, 6-oxo-1-heptyl, 1-methyl-4-oxo-pentyl, 4-methylthio-1-butyl, 5-methyl-4-oxo-hexyl, 1-ethyl-4-oxo-pentyl, N-(2-hydroxyethyl)phthalimido, 2-(N-acetyl-N-methyl)aminoethyl, 2-(N-formyl-N-methyl)aminoethyl, 2-methyl-5-oxo-hexyl, 1-dimethyl-4-oxo-pentyl, 4-oxo-1-octyl, 4-oxo-1-tetradecyl, 4-oxo-1-eicosamyl, and 3-(N-tertbutylcarboxamido)-1-propyl.

12. The method according to claim 1, wherein said modification group dissociates during the initial denaturation step of said amplification.

13. The method of claim 1, wherein said modification group partially or entirely dissociates at a temperature between about 37°

C.-95°

C.

14. The method of claim 1, wherein said modified oligonucleotide primer comprises a phosphotriester internucleotide linkage.

15. The method of claim 1, wherein said modification group impairs hybridization of the oligonucleotide primer to a nucleic acid sequence.

16. The method of claim 1, wherein said modification group impairs polymerase extension of said modified oligonucleotide primer.

17. The method of claim 1, wherein said modified oligonucleotide primer comprises two or more modification groups at the n, n-1, n-2, n-3, n-4, n-5 or n-6 position;

wherein n is the 3′

terminal internucleotide linkage.

18. The method of claim 1, wherein said modified oligonucleotide primer comprises two or more modification groups.

19. The method of claim 1, wherein t_1/2 of dissociation of said modification group is between about 0.1-120 minutes at 95°

C.

20. The method of claim 1, wherein the modification group dissociates between 50-95°

C.

21. The method of claim 1, wherein said nucleic acid amplification comprises a hot start polymerase.

22. The method of claim 1 wherein said modified oligonucleotide primer is at least 90% chirally pure.

23. The method of claim 1, wherein said oligonucleotide primer comprises a detectable label.

27. The method of claim 1, wherein said amplifying comprises polymerase chain reaction (PCR).

28. The method of claim 27, wherein said PCR comprises multiplex PCR.

29. The method of claim 28, wherein a first target comprises amplifying using unmodified primers and a second target is amplified using modified primers.

30. The method of claim 28, wherein a first target comprises amplifying using one or more primers with a first modification group and a second target is amplified using one or more modified primers with a second modification group, wherein said first and second modification groups are different.

31. The method of claim 1, wherein said amplifying comprises reverse transcriptase (RT).

32. The method of claim 31, wherein said modification group dissociates at about 37-70°

C.

34. The method of claim 31, wherein said modification group dissociates at 42°

C.

35. The method of claim 1, wherein said amplifying comprises subsequent enzymatic reactions in a single tube.

36. The method of claim 35, wherein said subsequent enzymatic reactions comprise reverse transcriptase (RT) as a first enzymatic reaction and polymerase chain reaction (PCR) as a second enzymatic reaction.

37. The method of claim 36, wherein said RT reaction comprises amplifying with one or more unmodified primers and said PCR reaction comprises amplifying with one or more modified primers.

38. The method of claim 36, wherein said RT reaction comprises amplifying with one or more of a first modified primer and said PCR reaction comprises amplifying with one or more of a second modified primer.

39. The method of claim 38, wherein said one or more of a first modified primer comprises a modification group that dissociates at about 42°

C., and wherein said one or more of a second modified primer comprises a modification group that dissociates at about 95°

C.

24. An oligonucleotide primer for nucleic acid amplification comprising:

a nucleic acid sequence;

wherein said nucleic acid sequence has one or more modification groups; and

wherein said modification group comprises one or more thermally cleavable groups.

25. The oligonucleotide primer of claim 24, wherein said modified oligonucleotide primer is between about 6 and 70 nucleotides in length.

26. A kit for nucleic acid amplification comprising one or more modified oligonucleotides wherein said modified oligonucleotides comprises one or more thermally cleavable groups.