Methods and compositions for multiple displacement amplification of nucleic acids
First Claim
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.
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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.
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Citations
16 Claims
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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.
- 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′
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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. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
- a plurality enzymes having enzyme activity in a mixture, wherein said enzymes comprise Phi29 polymerase, DNA polymerase I, and pyrophosphatase;
Specification