Method for multiplexed nucleic acid patch polymerase chain reaction
First Claim
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1. 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 an upstream primer and a downstream primer to the at least two nucleic acid sequences to be amplified;
ii) amplifying the at least two nucleic acid sequences so as to create amplicons; and
iii) after amplifying, removing the upstream and downstream primer sequences from the amplicons of step ii);
b) annealing an upstream and a downstream nucleic acid patch to each of the amplicons of step iii), 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 the associated amplicon; and
d) amplifying the ligated nucleic acid sequences of step (c).
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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.
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Citations
31 Claims
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1. A method of amplifying at least two different nucleic acid sequences, the method comprising:
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a) creating known 5′ and
3′
ends of at least two nucleic acid sequences by;i) annealing an upstream primer and a downstream primer to the at least two nucleic acid sequences to be amplified; ii) amplifying the at least two nucleic acid sequences so as to create amplicons; and iii) after amplifying, removing the upstream and downstream primer sequences from the amplicons of step ii); b) annealing an upstream and a downstream nucleic acid patch to each of the amplicons of step iii), 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 the associated amplicon; and d) amplifying the ligated nucleic acid sequences of step (c). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of amplifying one or more than one unique nucleic acid sequences, the method comprising:
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a. annealing an upstream primer and a downstream primer to each unique nucleic acid sequence, wherein the upstream primer and the downstream primer comprise uracil instead of thymine; b. amplifying each nucleic acid sequence so as to create amplicons of the nucleic acid sequence; c. removing the upstream and downstream primer sequences from the amplicons of step (b) by contacting the amplicons with a uracil DNA glycosylase, an endonuclease, and an exonuclease; d. annealing upstream and downstream nucleic acid patches to each unique amplicon of step (c), and annealing an upstream universal primer to the upstream patch of each unique amplicon, and a downstream universal primer to the downstream patch of each unique amplicon, wherein the downstream universal primer comprises a protecting group; e. ligating the upstream universal primer and the downstream universal primer to each unique amplicon; f. degrading non-specific amplicons of step (e); and g. amplifying the amplicons of step (f). - View Dependent Claims (16, 17)
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18. A method of amplifying at least two different bisulfite treated nucleic acid sequences, the method comprising:
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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; and d) amplifying the nucleic acid sequences of step (c). - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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Specification