Method for polynucleotide amplification

• US 6,482,590 B1
• Filed: 11/06/1997
• Issued: 11/19/2002
• Est. Priority Date: 12/20/1996
• Status: Expired due to Fees

• Current Assignee: AVENTIS BEHRING GMBH (CSL Limited)
• Original Assignee: AVENTIS BEHRING GMBH (CSL Limited)
• Inventors: Kurn, Nurith, Ullman, Edwin F., Lishanski, Alla
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/965,492
• Time in Patent Office: 1,839 Days
• Field of Search: 435/91.2, 435/91.1, 536/23.1
• US Class Current: 435/6.12
• 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 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. Selective extension is achieved by controlling the concentration of the oligonucleotide primer by forming the oligonucleotide in situ by degrading the 3′-end of a modified oligonucleotide. The method comprises providing in combination the mixture of polynucleotides and the modified oligonucleotide having a 3′-end that is not extendable along any polynucleotide and extending the oligonucleotide primer selectively along the specific target polynucleotide sequence by controlling the degradation of the 3′-end of the modified oligonucleotide. In this way extension of the oligonucleotide primer along polynucleotides other than the specific target polynucleotide sequence is substantially reduced or avoided. In another aspect the invention is an improvement in a method for amplifying a target polynucleotide sequence. The method comprises combining the target polynucleotide sequence with reagents for amplifying the target polynucleotide sequence and subjecting the combination to conditions wherein the target polynucleotide sequence is amplified. The reagents comprise an oligonucleotide primer and a polymerase. The improvement comprises deriving the oligonucleotide primer from a modified oligonucleotide having a portion that hybridizes to the target polynucleotide sequence except for the 3′-end thereof, which has at least one nucleotide analog that is incapable of hybridizing to a polynucleotide. Kits for carrying out the above methods are also disclosed.

Claims

1. A method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides, said method comprising:

(a) providing in combination said mixture and a modified oligonucleotide having a 3′

-end that is inefficiently extendable in a template dependent manner along any polynucleotide sequence; and

(b) controlling the template dependent extension of said oligonucleotide primer along said specific target polynucleotide sequence by controlling the degradation of said 3′

-end of said modified oligonucleotide thereby producing said oligonucleotide primer.

2. The method of claim 1 wherein said degradation is enzymatically catalyzed.

3. The method of claim 1 wherein said modified oligonucleotide comprises one or more nucleotide analogs at said 3′

-end that inhibit chain extension of said modified oligonucleotide along all polynucleotides in said mixture.

4. The method of claim 3 wherein said one or more nucleotide analogs hybridize inefficiently to any normal nucleotide in a polynucleotide.

5. In a template dependent method for amplifying a target polynucleotide sequence, said method comprising combining said target polynucleotide sequence with reagents for amplifying said target polynucleotide sequence and subjecting said combination to conditions wherein said target polynucleotide sequence is amplified in a template dependent manner, said reagents comprising an oligonucleotide primer and a polymerase, the improvement which comprises said oligonucleotide prime being derived from a modified oligonucleotide having a portion that hybridizes to said target polynucleotide sequence except for the 3′

-end thereof, said 3′

-end having at least one nucleotide analog that is substantially incapable of hybridizing to a polynucleotide, said 3′

-end being degradable to provide said oligonucleotide primer.

6. The method of claim 5 wherein said modified oligonucleotide has a 3′

-end that comprises at least two unnatural nucleotides that do not hybridize to said target polynucleotide sequence.

7. The method of claim 5 wherein said at least one nucleotide analogue is a natural nucleotide having a chemical modification that is removeable.

8. The method of claim 5 wherein said nucleotide analog is removable by an enzyme having 3′

to 5′

exonuclease activity.

9. The method of claim 7 wherein said chemical modification is selected from the group consisting of esters, glycosides, sulfates and amides.

10. A method for amplifying a target polynucleotide sequence, which comprises:

(a) providing in combination (i) said target polynucleotide sequence and (ii) reagents for conducting a template dependent amplification of said target polynucleotide sequence, said reagents comprising a polymerase, nucleoside triphosphates and a modified oligonucleotide comprising at least one nucleotide analog at the 3′

-end thereof, said nucleotide analog being substantially non-hyridizable to any polynucleotide sequence and being removable by a 3′

to 5′

exonuclease at a rate that is slower than for a natural nucleotide.

(b) subjecting said combination to conditions for amplifying said target polynucleotide sequence in a template dependent manner, wherein said nucleotide analog is removed from said modified oligonucleotide to give an oligonucleotide primer, which is then extended along said target polynucleotide sequence to produce an extended oligonucleotide primer.

11. The method of claim 10 which comprises detecting said target polynucleotide sequence by detecting said extended oligonucleotide primer.

12. The method of claim 10 wherein said reagents comprise a second modified oligonucleotide that is degradable to a second oligonucleotide primer.

13. The method of claim 10 wherein said nucleotide analog has a 3′

-hydroxyl group.

14. The method of claim 10 wherein said nucleotide analog is a natural nucleotide having a chemical modification that is removable.

15. The method of claim 10 wherein said nucleotide analog is removable by an enzyme having 3′

to 5′

exonuclease activity.

16. The method of claim 14 wherein said chemical modification is selected from the group consisting of esters, glycosides, sulfates and amides.

17. The method of claim 10 wherein said nucleotide analog is at the 3′

-terminus of said modified oligonucleotide.

18. The method of claim 10 wherein said nucleotide analog is within one nucleotide of the 3′

-terminus of said modified oligonucleotide.

19. The method of claim 10 wherein said nucleotide analog is attached directly to a sequence of 12 to 50 nucleotides of said modified oligonucleotide that hybridize to said target polynucleotide sequence.

20. The method of claim 10 wherein said nucleotide analog is comprised of an unnatural base selected from the group consisting of adenine modified at the 6-amino group, guanine modified at the 6-oxygen, thymine modified at the 4-oxygen, purines modified at the ring nitrogens at the 5-position, pyrimidines modified at the ring nitrogens at the 3-position, guanine modified by removal of the 2-amino group and cytosine modified by the removal of the 4-amino group.

21. The method of claim 10 wherein said target polynucleotide is DNA.

22. In a template dependent method for amplifying a target polynucleotide sequence, said method comprising combining said target polynucleotide sequence with reagents for amplifying said target polynucleotide sequence and subjecting said combination to conditions wherein said target polynucleotide sequence is amplified in a template dependent manner, said reagents comprising an oligonucleotide primer and a polymerase, the improvement which comprises said oligonucleotide primer being derived from a modified oligonucleotide having a portion that hybridizes to said target polynucleotide sequence except for the 3′

-end thereof, said 3′

-end being degradable at a rate of degradation of said oligonucleotide primer hybridized to said target polynucleotide sequence is slow relative to the rate of degradation of said 3′

-end of non-hybridized oligonucleotide primer.

23. In a method for forming multiple copies of a target polynucleotide sequence, said method comprising:

(a) hybridizing to the 3′

-end of said target sequence a first oligonucleotide primer (“

first primer”

),

(b) extending in a template dependent manner in the presence of a polymerase, said first primer along at least said target sequence, said first primer being capable of hybridizing to, and being extended along, (1) said extended first primer or (2) an extended second oligonucleotide primer (“

second primer”

) wherein said extended second primer results from the extension of a second primer capable of hybridizing to and extending along a polynucleotide that is complementary (complementary polynucleotide) to said target sequence,

(c) dissociating said extended first primer from said target sequence,

(d) hybridizing, to the 3′

-end of said extended first primer, said first or said second primer,

(e) extending said first or said second primer along said extended first primer,

(f) dissociating said extended first primer or said extended second primer from said extended first primer,

(g) hybridizing, to the 3′

-end of said extended first or second primer, said first primer, and

(h) repeating steps (e)-(g),

the improvement comprising said first oligonucleotide primer being derived from a modified oligonucleotide having a portion that hybridizes to said target polynucleotide sequence except for the 3′

-end thereof, said 3′

-end having at least one nucleotide analog, said nucleotide analog comprising at least one natural nucleotide having a chemical modification that is removable, said nucleotide analog further being incapable of hybridizing to a polynucleotide, whereby said extension steps (b) and (h) are controlled by the removal of said chemical modification or said analog.

24. The method of claim 23 wherein said modified oligonucleotide has a 3′

-end that comprises at least two unnatural nucleotides that do not hybridize to said target polynucleotide sequence.

25. The method of claim 23 wherein said nucleotide analog is removable by an enzyme having 3′

to 5′

exonuclease activity.

26. The method of claim 23 wherein said chemical modification is selected from the group consisting of esters, glycosides, sulfates and amides.

27. The method of claim 23 wherein the presence of said extended first primer and/or said extended second primer is detected and related to the presence of said target polynucleotide.

28. The method of claim 23 wherein the repeating of steps (e)-(g) is achieved by repeated temperature cycling.

29. The method of claim 28 wherein temperature cycling is repeated at least 3 times.

30. The method of claim 23 wherein said target polynucleotide is DNA.

31. The method of claim 23 wherein said extending is carried out in the presence of nucleoside triphosphates.

32. The method of claim 23 wherein said first and said second primers are different.

33. The method of claim 23 wherein said first and said second primers are different and said extended first primer is a template for said second primer and said extended second primer is a template for said first primer.

34. A method for controlling the concentration of an oligonucleotide primer in a reaction medium for conducting a reaction in which said oligonucleotide primer is extended along a polynucleotide template, said method comprising:

(a) providing in said reaction medium, together with reactants for extending said oligonucleotide primer along said polynucleotide template in a template dependent manner, a modified oligonucleotide having a degradable 3′

-end, said modified oligonucleotide being substantially incapable of being extended along said polynucleotide template, wherein the degradation of said 3′

-end results in the formation of said oligonucleotide primer and

(b) subjecting said reaction medium to controlled conditions for degrading said 3′

-end of said modified oligonucleotide thereby forming said oligonucleotide primer.

35. The method of claim 34 wherein said 3′

-end comprises at least one nucleotide analog that is incapable of hybridizing with a natural nucleotide.