Methods and compositions for multiple displacement amplification of nucleic acids

Patent metadata

Field	Value
Publication number	US-9598724-B2
Application number	US-60264108-A
Country	US
Kind code	B2
Filing date	May 30, 2008
Priority date	Jun 1, 2007
Publication date	Mar 21, 2017
Grant date	Mar 21, 2017

Abstract

Disclosed are methods for multiple displacement amplification of a nucleic acid sequence in a sample. The nucleic acid is contacted with a reaction mixture that includes a set of oligonucleotide primers and a plurality of polymerase enzymes. The reaction mixture is subjected to conditions under which the nucleic acid sequence is amplified to produce an amplification product in a multiple displacement amplification reaction. Also disclosed are kits containing a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases. At least some of the individual members of the primers have one or more ribose modifications that stabilize or lock the ribose ring in a 3′ -endo conformation. At least some of the primers have one or more universal nucleobases.

First claim

What is claimed is:

1. A reaction mixture for multiple displacement amplification of a nucleic acid sample consisting of: a set of oligonucleotide primers wherein at least one primer of said set of oligonucleotide primers comprises one or more universal nucleobases, and at least one primer of said set of oligonucleotide primers comprises one or more phosphorothioate linkages, or one or more nucleotide residues with a ribose modification that stabilizes a 3′-endo conformation of said ribose ring or locks said ribose ring in said 3′-endo conformation or a combination thereof; a plurality of enzymes having enzyme activity, wherein said enzymes comprise Phi29 polymerase, DNA polymerase I, and pyrophosphatase; a set of natural deoxynucleotide triphosphates; and one or more compatible solutes.

2. A kit for multiple displacement amplification of a nucleic acid sample comprising: a plurality enzymes having enzyme activity in a mixture, wherein said enzymes comprise Phi29 polymerase, DNA polymerase I, and pyrophosphatase; a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases wherein at least some individual primers of said set of oligonucleotide primers comprise: one or more ribose modifications that stabilize a 3′-endo conformation of said ribose ring or lock said ribose ring in said 3′-endo conformation; and one or more universal nucleobases.

3. The kit of claim 2 wherein said individual primers further comprise one or more phosphorothioate linkages.

4. The kit of claim 2 wherein said set of oligonucleotide primers include at least some heptamer primers.

5. The kit of claim 2 wherein said universal nucleobase is inosine.

6. The kit of claim 4 wherein said heptamers have inosine residues at the 6 th and 7 th positions.

7. The kit of claim 4 wherein said heptamers have one or more phosphorothioate linkages.

8. The kit of claim 7 wherein said phosphorothioate linkages are located between the 5 th and 6 th residue and between the 6 th and 7 th residue of said heptamers.

9. The kit of claim 2 wherein said ribose modifications include a 2′ to 4′ ribose bridge.

10. The kit of claim 2 wherein said modifications are located at the 2 nd and 5 th positions of said primers.

11. The kit of claim 2 further comprising one or more compatible solutes.

12. The kit of claim 11 wherein said compatible solutes comprise betaine.

13. The kit of claim 11 wherein said compatible solutes comprise trehalose.

14. The kit of claim 11 wherein said compatible solutes comprise betaine and trehalose.

15. The kit of claim 2 further comprising a set of natural deoxynucleotide triphosphates.

16. The kit of claim 2 further comprising instructions for performing said multiple displacement amplification of said nucleic acid sample.

Assignees

• Duncan David D
• Eshoo Mark W
• Ibis Biosciences Inc

Inventors

• Duncan David D
• Eshoo Mark W

Classifications

• C12Q1/6846 Primary

  Common amplification features · CPC title

• C12Q2525/101

  incorporating non-naturally occurring nucleotides, e.g. inosine · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title