Method of whole genome amplification with reduced artifact production
First Claim
1. A method of amplifying nucleic acids, the method comprising,incubating nucleic acids comprising target sequences at an elevated temperature in the presence of a thermolabile nucleic acid polymerase having strand displacement activity, an additive, and a set of primers, under conditions promoting replication of the nucleic acids, wherein replication of the nucleic acids results in replicated strands, wherein during replication at least one of the replicated nucleic acid strands is displaced by strand displacement replication of another replicated strand, wherein replication is conducted at a constant temperature,wherein formation of replicated strands from the target sequences is favored over formation of replicated strands from non-target sequences.
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Abstract
Disclosed are compositions and methods for amplification of nucleic acid sequences of interest with greater efficiency and fidelity. The disclosed method relates to isothermal amplification techniques, such as Multiple Displacement Amplification (MDA), where the generation of DNA artifacts is decreased or eliminated. Generally, this can be accomplished by carrying out the reaction at elevated temperature. In particularly useful embodiments of the method, sugars and/or other additives can be used to stabilized the polymerase at high temperature. It has been discovered that generation of high molecular weight artifacts, in an isothermal amplification procedure, is substantially reduced or eliminated while still allowing the desired amplification of input DNA by carrying out the reaction at a higher temperature and, optionally, in the presence of one or more additives. It also has been discovered that isothermal amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed at a higher temperature and, optionally, in the presence of one or more additives.
119 Citations
28 Claims
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1. A method of amplifying nucleic acids, the method comprising,
incubating nucleic acids comprising target sequences at an elevated temperature in the presence of a thermolabile nucleic acid polymerase having strand displacement activity, an additive, and a set of primers, under conditions promoting replication of the nucleic acids, wherein replication of the nucleic acids results in replicated strands, wherein during replication at least one of the replicated nucleic acid strands is displaced by strand displacement replication of another replicated strand, wherein replication is conducted at a constant temperature, wherein formation of replicated strands from the target sequences is favored over formation of replicated strands from non-target sequences.
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26. A method of amplifying a whole genome, the method comprising,
exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, reducing the pH of the cell lysate to form a stabilized cell lysate, and incubating stabilized cell lysate at an elevated temperature in the presence of a thermolabile nucleic acid polymerase having strand displacement activity, an additive, and a set of primers, under conditions promoting replication of the nucleic acids, wherein replication of the nucleic acids results in replicated strands, wherein during replication at least one of the replicated nucleic acid strands is displaced by strand displacement replication of another replicated strand, wherein formation of replicated strands from the target sequence is favored over formation of replicated strands from non-target sequences.
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27. A method of performing strand displacement nucleic acid synthesis at an elevated temperature, the method comprising,
mixing thermolabile nucleic acid polymerase having strand-displacement activity, nucleic acids comprising target sequences, a set of primers, and an additive and incubating at an elevated temperature and under conditions favoring hybridization of the primers to the target sequences and extension of the primers by the addition of nucleotides sequentially to the 3′ - end of the primer in a template-dependent manner, wherein the extension results in replication of the target sequences.
Specification
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Current AssigneeQiagen GmbH (Qiagen NV)
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Original AssigneeQiagen GmbH (Qiagen NV)
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InventorsKumar, Gyanendra
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Primary Examiner(s)Babic; Christopher M.
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Application NumberUS10/456,056Publication NumberTime in Patent Office2,923 DaysField of SearchNoneUS Class Current435/6.12CPC Class CodesC12Q 1/6844 Nucleic acid amplification ...C12Q 1/6848 characterised by the means ...C12Q 2527/101 TemperatureC12Q 2531/119 Strand displacement amplifi...
