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US 8,936,912 B2Granted
Method for multiplexed nucleic acid patch polymerase chain reaction
AssigneeWashington University
InventorsRobi M. Mitra, Katherine E. Varley
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Abstract
The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.
Claims25
A method of amplifying at least two different nucleic acid sequences, the method comprising:
- a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by restriction enzyme digestion,
- b) combining, in a single reaction, a plurality of upstream and downstream nucleic acid patches, a plurality of upstream and downstream universal primers, and the nucleic acid sequences of known ends from step (a), wherein the downstream universal primers comprise a protecting group;
- c) performing at least two annealing cycles, wherein each cycle comprises denaturing the nucleic acid sequences of known ends from step (a), and cooling the reaction to anneal an upstream and downstream nucleic acid patch to a nucleic acid sequence of step (a) and an upstream universal primer to an upstream patch and a downstream universal primer to a downstream patch;
- d) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
- and
- e) amplifying the nucleic acid sequences of step (d).
3 more elements
A method of amplifying at least two different nucleic acid sequences, the method comprising:
- a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by restriction enzyme digestion;
- b) combining, in a single reaction, a plurality of upstream and downstream nucleic acid patches, a plurality of upstream and downstream universal primers, and the nucleic acid sequences of known ends from step (a);
- c) performing at least two annealing cycles, wherein each cycle comprises denaturing the nucleic acid sequences of known ends from step (a), and cooling the reaction to anneal an upstream and downstream nucleic acid patch to a nucleic acid sequence of step (a) and an upstream universal primer to an upstream patch and a downstream universal primer to a downstream patch;
- d) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
- and
- e) amplifying the nucleic acid sequences of step (d) using an upstream and a downstream amplification primer, wherein the upstream amplification primer comprises a first universal sequence and a sequence corresponding to the upstream universal primer of step (b), and wherein the downstream amplification primer comprises a second universal sequence and a sequence corresponding to the downstream universal primer of step (b).
3 more elements
A method of amplifying at least two different nucleic acid sequences, the method comprising:
- a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by:
- i) single strand specific exonuclease enzyme digestion, or
- ii) triplex formation,
- b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of known ends from step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
- c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
- and
- d) amplifying the nucleic acid sequences of step (c).
4 more elements
A method of amplifying at least two different nucleic acid sequences, the method comprising:
- a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by:
- i) annealing oligonucleotides to the at least two nucleic acid sequences, wherein the oligonucleotides comprise a locus specific sequence and a Type IIS restriction enzyme recognition sequence, and
- ii) contacting the at least two nucleic acid sequences with a Type IIS restriction enzyme, under conditions suitable for the enzymes to cleave the at least two nucleic acid sequences creating known 5′ and 3′ ends,
- b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of known ends from step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
- c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
- and
- d) amplifying the nucleic acid sequences of step (c).
4 more elements
A method of amplifying at least two different nucleic acid sequences, the method comprising:
- a) creating known 5′ and 3′ ends of at least two nucleic acid sequences, such that at least three nucleic acid bases are known at both the 5′ and the 3′ ends,
- b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
- c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
- d) treating the nucleic acid sequences with bisulfite;
- and
- e) amplifying the nucleic acid sequences of step (d).
3 more elements
Dependencies are inferred from the claim text.