Selected amplification of polynucleotides
First Claim
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1. A method of forming closed double stranded DNA circles from a plurality of target polynucleotides in a sample, the method comprising the steps of:
- providing one or more selection oligonucleotides for each of the plurality of target polynucleotide so that each of such one or more selection oligonucleotides has a phosphorylated 5′
end and is capable of specifically annealing at the same time to two non-contiguous sites on its respective target polynucleotide whenever present in the sample to form a circular complex therewith comprising a free 5′
strand and a free 3′
strand; and
combining in a reaction mixture the sample, nucleoside triphosphates, and the selection oligonucleotides under conditions such that the following enzymatic activities are present;
(i) a 5′
flap endonuclease activity, (ii) a DNA polymcrasc lacking strand displacement activity, (iii) a 3′
single stranded exonuclease activity, and (iv) a ligase activity, wherein for each circular complex any free 3′
strand is digested to form an extendable duplex that is extended by the DNA polymerase activity to the free 5′
strand of the circular complex, any free 5′
strand of the circular complex adjacent to an extended extendable duplex is cleaved to form a first nick, the annealed selection oligonucleotide is extended from a 3′
end along the circular complex to a 5′
end of the selection oligonucleotide to form a second nick, and the first and second nicks are ligated to form a closed double stranded DNA circle.
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Abstract
The invention provides methods and compositions for selectively amplifying one or more target polynucleotides in a sample. In one aspect, a plurality of selection oligonucleotides are provided that are capable of simultaneously annealing to separate regions of a target polynucleotide to form a complex that is enzymatically converted into a closed double stranded DNA circle that incorporates the sequence region between the two separate regions. Sequences that fail to form such complexes may be removed by nuclease digestion and the sequences of the remaining DNA circles may be amplified by a variety of techniques, such as rolling circle replication after nicking, PCR amplification after linearization, or the like.
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Citations
21 Claims
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1. A method of forming closed double stranded DNA circles from a plurality of target polynucleotides in a sample, the method comprising the steps of:
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providing one or more selection oligonucleotides for each of the plurality of target polynucleotide so that each of such one or more selection oligonucleotides has a phosphorylated 5′
end and is capable of specifically annealing at the same time to two non-contiguous sites on its respective target polynucleotide whenever present in the sample to form a circular complex therewith comprising a free 5′
strand and a free 3′
strand; andcombining in a reaction mixture the sample, nucleoside triphosphates, and the selection oligonucleotides under conditions such that the following enzymatic activities are present;
(i) a 5′
flap endonuclease activity, (ii) a DNA polymcrasc lacking strand displacement activity, (iii) a 3′
single stranded exonuclease activity, and (iv) a ligase activity, wherein for each circular complex any free 3′
strand is digested to form an extendable duplex that is extended by the DNA polymerase activity to the free 5′
strand of the circular complex, any free 5′
strand of the circular complex adjacent to an extended extendable duplex is cleaved to form a first nick, the annealed selection oligonucleotide is extended from a 3′
end along the circular complex to a 5′
end of the selection oligonucleotide to form a second nick, and the first and second nicks are ligated to form a closed double stranded DNA circle. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of amplifying a plurality of target polynucleotides in a sample, the method comprising the steps of:
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providing one or more selection oligonucleotides for each of the plurality of target polynucleotide so that each of such one or more selection oligonucleotides has a phosphorylated 5′
end and is capable of specifically annealing at the same time to two non-contiguous sites on its respective target polynucleotide whenever present in the sample to form a circular complex therewith comprising a free 5′
strand and a free 3′
strand;combining in a reaction mixture the sample, nucleoside triphosphatcs, and the selection oligonucleotides under conditions such that the following enzymatic activities are present;
(i) a 5′
flap endonuclease activity, (ii) a DNA polymerase lacking strand displacement activity, (iii) a 3′
single stranded exonuclease activity, and (iv) a ligase activity, wherein for each circular complex any free 3′
strand is digested to form an extendable duplex that is extended by the DNA polymerase activity to the free 5′
strand of the circular complex, any free 5′
strand of the circular complex adjacent to an extended extendable duplex is cleaved to form a first nick, the annealed selection oligonucleotide is extended from a 3′
end along the circular complex to a 5′
end of the selection oligonucleotide to form a second nick, and the first and second nicks are ligated to form a closed double stranded DNA circle;treating the reaction mixture with nucleases to destroy oligonucleotides and polynucleotides that are not closed double stranded DNA circles; linearizing the closed double stranded DNA circle to form a linear double stranded DNA; and amplifying the linear double stranded DNA in a polymerase chain reaction. - View Dependent Claims (14, 15, 16, 17, 18, 19)
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20. A method of forming closed double stranded DNA circles from a plurality of target polynucleotides in a sample, the method comprising the step of:
combining in a reaction mixture the sample, one or more selection oligonucleotides for each of the plurality of target polynucleotides and nucleoside triphosphates, each of the one or more selection oligonucleotides having a phosphorylated 5′
end and a nuclease resistant 3′
end and specifically annealing at the same time to two non-contiguous sites on its respective target polynucleotide whenever present in the sample to form a circular complex therewith comprising a free 5′
strand and a free 3′
strand, and the reaction mixture having conditions such that the following enzymatic activities are present;
(i) a 5′
flap endonuclease activity, (ii) a DNA polymerase lacking strand displacement activity. (iii) a 3′
single stranded exonuclease activity, and (iv) a ligase activity, such that for each circular complex any free 3′
strand is digested to form an extendable duplex that is extended by the DNA polymerase activity to the free 5′
strand of the circular complex, any free 5′
strand of the circular complex adjacent to an extended extendable duplex is cleaved to form a first nick, the annealed selection oligonucleotide is extended from a 3′
end along the circular complex to a 5′
end of the selection oligonucleotide to form a second nick, and the first and second nicks are ligated to form a closed double stranded DNA circle.- View Dependent Claims (21)
Specification
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Current AssigneeStephen C. Macevicz
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Original AssigneeStephen C. Macevicz
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InventorsMacevicz, Stephen C.
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Primary Examiner(s)Strzelecka, Teresa E
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Assistant Examiner(s)Pande, Suchira
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Application NumberUS11/564,329Publication NumberTime in Patent Office1,938 DaysField of SearchNoneUS Class Current435/91.2CPC Class CodesC12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6813 Hybridisation assays
