DNA amplification technology

• US 10,669,574 B2
• Filed: 07/10/2015
• Issued: 06/02/2020
• Est. Priority Date: 11/18/2008
• Status: Active Grant

• Current Assignee: Xcr Diagnostics, Inc.
• Original Assignee: Xcr Diagnostics, Inc.
• Inventors: Caplin, Brian, Green, Bryson
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US15/324,218
• Publication Number:
  US 20170198342A1
• Time in Patent Office: 1,789 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Methods and reagents suitable for conducing polymerase chain reaction are described. In particular, a nucleic acid amplification design strategy and thermal cycling profile to enable efficient amplification of multiple nucleic acid targets along with improved sensitivity is disclosed. The present disclosure also describes methods and devices for increasing the melting temperature (Tm) of a primer.

Claims

1. A nucleic acid complex, comprising a double-stranded target nucleic acid and a first oligonucleotide primer and a second oligonucleotide primer hybridized thereto,

wherein the first oligonucleotide primer comprises a 3′

end having a sequence substantially complementary to a binding site on the first strand of the target nucleic acid and a 5′

end substantially complementary to a binding site on the second strand of the target nucleic acid opposite to the binding she of the 3′

end of the first oligonucleotide primer;

wherein the second oligonucleotide primer comprises a 3′

end having a sequence substantially complementary to a binding site on the second strand of the target nucleic acid and a 5′

end substantially complementary to a binding site on the first strand of the target nucleic acid opposite to the binding site of the 3′

end of the second oligonucleotide primer;

wherein on the first strand, the binding site of the 3′

end of the first oligonucleotide primer is 3′

to the binding site of the 5′

end of the second oligonucleotide primer; and

wherein on the second strand, the binding site of the 3′

end of the second oligonucleotide primer is 3′

to the binding site of the 5′

end of the first oligonucleotide primer.

2. The nucleic acid complex of claim 1, wherein each oligonucleotide primer comprises a transition between the 3′

end and the 5′

end of said oligonucleotide primer.

3. The nucleic acid complex of claim 2, wherein the transition comprises a single nucleotide, a chain of carbons, a multifunctional moiety, modified nucleotides, modified backbones or a combination thereof.

4. The nucleic acid complex of claim 1, wherein the nucleic acid complex comprises a target nucleic acid sequence on the first strand of the target nucleic acid, wherein the target nucleic acid sequence comprises the binding site of the 3′

end of the first oligonucleotide primer, the binding site of the 5′

end of the second oligonucleotide primer, and the portion of the first strand between said two binding sites; and

wherein the Tm of the first oligonucleotide primer and the Tm of the second oligonucleotide primer are within at most 15°

C. of the Tm of the target nucleic acid sequence.

5. The nucleic acid complex of claim 1, wherein the Tms of the oligonucleotide primers are within at most 10°

C. of the Tm of the target nucleic acid sequence.

6. The nucleic acid complex of claim 1, wherein the Tms of the oligonucleotide primers are within at most 5°

C. of the Tm of the target nucleic acid sequence.

7. The nucleic acid complex of claim 1, wherein the Tms of the oligonucleotide primers are within at most 2.5°

C. of the Tm of the target nucleic acid sequence.

8. The nucleic acid complex of claim 1, wherein the Tms of the oligonucleotide primers are equal to the Tm of the target nucleic acid sequence.

9. The nucleic acid complex of claim 1, wherein at least one of the 5′

ends comprises nucleotide or backbone modifications to optimize annealing of the oligonucleotide primer to the binding site of said 5′

end.

10. The nucleic acid complex of claim 1, wherein at least one of the oligonucleotide primers comprises a sequence of cytosine nucleotides adjacent to a first sequence of guanosine nucleotides.

11. The nucleic acid complex of claim 10, wherein the number of nucleotides between the cytosine and guanosine nucleotides is less than 5.

12. The nucleic acid complex of claim 10, wherein the number of nucleotides between the cytosine and guanosine nucleotides is less than 4.

13. The nucleic acid complex of claim 10, wherein the number of nucleotides between the cytosine and guanosine nucleotides is less than 3.

14. The nucleic acid complex of claim 10, wherein the number of nucleotides between the cytosine and guanosine nucleotides is less than 2.

15. The nucleic acid complex of claim 10, wherein the number of nucleotides between the cytosine and guanosine nucleotides is 0.

16. The nucleic acid complex of claim 10, wherein the at least one oligonucleotide primer can form a Guanosine quadruplex structure.

17. The nucleic acid complex of claim 10, wherein the at least one oligonucleotide primer further comprises a second sequence of guanosine nucleotides adjacent to the first sequence of guanosine nucleotides.

18. The nucleic acid complex of claim 17, wherein the second sequence of guanosine nucleotides causes the primer to shift and form a Guanosine quadruplex structure.

19. A method for increasing the melting temperature (Tm) of oligonucleotide primers to be used in a polymerase chain reaction (PCR), comprising:

(i) selecting a double-stranded target nucleic acid;

(ii) designing a first oligonucleotide primer comprising a 3′

end substantially complementary to a binding site on the first strand of the target nucleic acid and a 5′

end substantially complementary to a binding site on the second strand of the target nucleic acid opposite to the binding site of the 3′

end of the first oligonucleotide primer;

(iii) designing a second oligonucleotide primer comprising a 3′

end substantially complementary to a binding site on the second strand of the target nucleic acid and a 5′

end substantially complementary to a binding site on the first strand of the target nucleic acid opposite to the binding site of the 3′

end of the second oligonucleotide primer;

wherein the first oligonucleotide primer and the second oligonucleotide primer form the nucleic acid complex of claim 1 when incubated with the double-stranded target nucleic acid;

wherein the double-stranded target nucleic acid contains a target nucleic acid sequence on its first strand that comprises the binding site of the 3′

end of the first oligonucleotide primer, the binding site of the 5′

end of the second oligonucleotide primer, and the portion of said first strand between said two binding sites; and

wherein the Tm of the first oligonucleotide primer and the Tm of the second oligonucleotide primer are within at most 15°

C. of the Tm of said target nucleic acid sequence; and

(iv) synthesizing said first and second oligonucleotide primers.

20. The method of claim 19, wherein the Tms of the oligonucleotide primers are within at most 10°

C. of the Tm of the target nucleic acid sequence.

21. The method of claim 19, wherein the Tms of the oligonucleotide, primers are within at most 5°

C. of the Tm of the target nucleic acid sequence.

22. The method of claim 19, wherein the Tms of the oligonucleotide primers are within at most 2.5°

C. of the Tm of the target nucleic acid sequence.

23. The method of claim 19, wherein the Tms of the oligonucleotide primers are equal to the Tm of the target nucleic acid sequence.

24. A method for nucleic acid sequence amplification, comprising:

(i) generating a solution comprising the nucleic acid complex of claim 1; and

(ii) amplifying a target nucleic acid sequence in said nucleic acid complex by subjecting the solution to thermal cycling;

wherein the nucleic acid complex of claim 1 is generated by a process that includes incubating the double-stranded target nucleic acid, the first olignucleotide primer, and the second olignucleotide primer at a temperature selected to minimize non-target nucleic acid sequence denaturation and maximize target nucleic acid sequence denaturation.