Complexity management of genomic DNA
First Claim
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1. A method of reducing the complexity of a first nucleic acid sample to produce a second nucleic acid sample comprising:
- fragmenting a first nucleic acid sample to produce double-stranded fragments;
ligating one or more adaptors to the double-stranded fragments;
digesting the adaptor ligated double-stranded fragments with an exonuclease that catalyzes the removal of mononucleotides from the 3′
termini of duplex DNA but not the 5′
termini or an exonuclease that catalyzes the removal of mononucleotides from the 5′
termini of duplex DNA but not the 3′
termini, wherein said exonuclease preferentially degrades duplex DNA, to produce single-stranded half molecules;
adding a homopolymeric tail to the 3′
end of the single stranded half molecules, hybridizing a primer to the homopolymeric tail, and extending the primer to generate double-stranded half fragments from the single-stranded half molecules;
amplifying a plurality of the double-stranded fragments by a polymerase chain reaction (PCR), and modulating the size of the amplified fragments by varying one or more reaction conditions or reagents to reduce the complexity of the first nucleic acid sample.
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Abstract
The presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for size fractionation. In a preferred embodiment, size fractionation can be accomplished by varying conditions or reagents of a PCR reaction to amplify fragments of specific size ranges. The invention further provides for analysis of the above sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.
87 Citations
43 Claims
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1. A method of reducing the complexity of a first nucleic acid sample to produce a second nucleic acid sample comprising:
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fragmenting a first nucleic acid sample to produce double-stranded fragments;
ligating one or more adaptors to the double-stranded fragments;
digesting the adaptor ligated double-stranded fragments with an exonuclease that catalyzes the removal of mononucleotides from the 3′
termini of duplex DNA but not the 5′
termini or an exonuclease that catalyzes the removal of mononucleotides from the 5′
termini of duplex DNA but not the 3′
termini, wherein said exonuclease preferentially degrades duplex DNA, to produce single-stranded half molecules;
adding a homopolymeric tail to the 3′
end of the single stranded half molecules, hybridizing a primer to the homopolymeric tail, and extending the primer to generate double-stranded half fragments from the single-stranded half molecules;
amplifying a plurality of the double-stranded fragments by a polymerase chain reaction (PCR), and modulating the size of the amplified fragments by varying one or more reaction conditions or reagents to reduce the complexity of the first nucleic acid sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42)
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37. A method ofreducing the complexity of a first nucleic acid sample to produce a second nucleic acid sample whereby the second nucleic acid sample is obtainable by:
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fragmenting the first nucleic acid sample to produce double-stranded fragments;
ligating adaptor sequences to both ends of the double-stranded fragments such that the 5′ and
3′
ends of the adaptor-ligated double-stranded fragments are complementary to one another;
digesting the adaptor ligated double-stranded fragments with an exonuclease that catalyzes the removal of mononucleotides from the 3′
termini of duplex DNA but not the 5′
termini or an exonuclease that catalyzes the removal of mononucleotides from the 5′
termini of duplex DNA but not the 3′
termini, wherein said exonuclease peferentially degrades duplex DNA, to produce single stranded half molecules;
adding a homopolymeric tail to the 3′
end of the single stranded half molecules, hybridizing a primer to the homopolymeric tail, and extending the primer to generate double stranded half fragments from the single stranded half molecules; and
,amplifying a subset of the double-stranded half fragments by a polymerase chain reaction (PCR) wherein a subset of fragments of a specific size range are preferentially amplified by varying the PCR primer concentration. - View Dependent Claims (38, 39, 40, 41, 43)
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Specification
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Current AssigneeAffymetrix, Inc. (Thermo Fisher Scientific Incorporated)
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Original AssigneeAffymetrix, Inc. (Thermo Fisher Scientific Incorporated)
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InventorsSu, Xing
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Primary Examiner(s)Fredman, Jeffrey
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Application NumberUS10/013,598Publication NumberTime in Patent Office1,205 DaysField of Search435/6, 435/91.2, 435/91.21, 435/18, 536/23.1US Class Current435/6.12CPC Class CodesC12Q 1/6855 Ligating adaptorsC12Q 2527/113 TimeC12Q 2527/143 Concentration of primer or ...
