Compositions and methods for representational selection of nucleic acids from complex mixtures using hybridization
First Claim
1. A method of selecting a representational sample of nucleic acid sequences from a complex mixture, comprising:
- (a) providing a complex mixture of nucleic acids comprising genomic DNA sequence having a complexity of at least 1.7 Gbp;
(b) providing a population of solid support-attached capture probes, said population comprising nucleic acids complementary to nucleic acids comprising a predetermined portion of the nucleic acid sequences collectively present in said complex mixture;
(c) contacting said complex mixture of nucleic acids with said population of solid support-attached capture probes under conditions sufficient for hybridization of said nucleic acids with said capture probes, wherein the capture probes are randomly located on the solid support;
(d) removing unhybridized nucleic acids from the solid support to separate desired sequences from undesired sequences within the complex mixture;
thereby selecting a representational sample of nucleic acids having a complexity of at least 0.001% and at most 49% of said complex mixture, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture;
(e) amplifying the nucleic acids in the representational sample to generate an amplified representational sample; and
(f) performing a sequence analysis of the amplified representational sample.
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Abstract
The invention provides a method of selecting a representational sample of nucleic acid sequences from a complex mixture. The method includes: (a) contacting a complex mixture of nucleic acids under conditions sufficient for hybridization with a population of capture probes complementary to one or more nucleic acids comprising a predetermined portion of the sequence collectively present in the complex mixture to form hybridization complexes of the one or more nucleic acids with the population of probes, the population of capture probes being attached to a solid support, and (b) removing unhybridized nucleic acids to select a representational sample of nucleic acids having a complexity of less than 10% but more than 0.001% of the complex mixture, wherein the representational sample comprises a nucleic acid copy having a proportion of each sequence in the copy relative to all other sequences in the copy substantially the same as the proportions of the sequences in the predetermined portion of one or more nucleic acids within the complex mixture. A method of selecting a representational sample of genomic sequences from a complete genome also is provided. The invention further provides a nucleic acid population that includes a representational sample having a complexity of less than 10% but more than 0.001% of a complex mixture, the representational sample comprising a nucleic acid copy having a proportion of each sequence in the copy relative to all other sequences in the copy substantially the same as the proportions of sequences in a predetermined portion of a sequence collectively present in one or more nucleic acids within the complex mixture.
37 Citations
20 Claims
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1. A method of selecting a representational sample of nucleic acid sequences from a complex mixture, comprising:
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(a) providing a complex mixture of nucleic acids comprising genomic DNA sequence having a complexity of at least 1.7 Gbp; (b) providing a population of solid support-attached capture probes, said population comprising nucleic acids complementary to nucleic acids comprising a predetermined portion of the nucleic acid sequences collectively present in said complex mixture; (c) contacting said complex mixture of nucleic acids with said population of solid support-attached capture probes under conditions sufficient for hybridization of said nucleic acids with said capture probes, wherein the capture probes are randomly located on the solid support; (d) removing unhybridized nucleic acids from the solid support to separate desired sequences from undesired sequences within the complex mixture;
thereby selecting a representational sample of nucleic acids having a complexity of at least 0.001% and at most 49% of said complex mixture, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture;(e) amplifying the nucleic acids in the representational sample to generate an amplified representational sample; and (f) performing a sequence analysis of the amplified representational sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of analyzing a complex mixture of nucleic acids comprising:
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(a) providing a complex mixture of nucleic acids comprising genomic DNA sequence having a complexity of at least 1.7 Gbp; (b) providing a population of solid support-attached capture probes, said population comprising nucleic acids complementary to nucleic acids comprising a predetermined portion of the nucleic acid sequences collectively present in said complex mixture; (c) contacting said complex mixture of nucleic acids with said population of solid support-attached capture probes under conditions sufficient for hybridization of said nucleic acids with said capture probes, wherein the capture probes are randomly located on the solid support, (d) removing unhybridized nucleic acids from the solid support to separate desired sequences from undesired sequences within the complex mixture;
thereby selecting a representational sample of nucleic acids having a complexity of at least 0.001% and at most 49% of said complex mixture, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture;(e) amplifying the nucleic acids in the representational sample to generate an amplified representational sample; and (f) detecting individual nucleic acids in said amplified representational sample to determine a sequence characteristic of said predetermined portion of the nucleic acid sequences collectively present in said complex mixture. - View Dependent Claims (16, 17, 18, 19, 20)
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Specification
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Current AssigneeIllumina Incorporated
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Original AssigneeIllumina Incorporated
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InventorsStuelpnagel, John R., Barker, David L., Velarde, Jorge Jr., Barnard, Steven M., Graige, Michael
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Primary Examiner(s)Martinell, James
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Application NumberUS14/046,859Publication NumberTime in Patent Office445 DaysField of SearchNoneUS Class Current435/6.11CPC Class CodesC12N 15/1072 Differential gene expressio...C12Q 1/6837 using probe arrays or probe...C12Q 2565/515 characterised by the intera...
