High throughput screening of mutagenized populations
First Claim
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1. A method for the detection of a mutation in a target sequence in one or more members of a mutagenized population, comprising:
- (a) providing a plurality of pools of amplification products each comprising the target sequence amplified from genomic DNA of a subset of the one or more members of the mutagenized population, wherein each pool of the amplification products shares a unique pool identifier;
(b) determining the nucleotide sequence of the amplification products using high throughput sequencing; and
(c) identifying mutations by clustering/aligning the sequences of the amplified products without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis, and identifying the one or more members carrying the mutation using the pool identifier.
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Abstract
Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.
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Citations
20 Claims
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1. A method for the detection of a mutation in a target sequence in one or more members of a mutagenized population, comprising:
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(a) providing a plurality of pools of amplification products each comprising the target sequence amplified from genomic DNA of a subset of the one or more members of the mutagenized population, wherein each pool of the amplification products shares a unique pool identifier; (b) determining the nucleotide sequence of the amplification products using high throughput sequencing; and (c) identifying mutations by clustering/aligning the sequences of the amplified products without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis, and identifying the one or more members carrying the mutation using the pool identifier. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification