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 a member of a mutagenized population, comprising the steps of:
- (a) isolating genomic DNA of each member of the mutagenized population to provide DNA samples of each population member;
(b) pooling the DNA obtained in step (a);
(c) amplifying the target sequence with a pair of primers from the DNA pools to obtain amplification products;
(d) pooling the amplification products of step (c) to create a library of amplification products;
(e) determining the nucleotide sequence of the amplification products or fragments of step (d) using high throughput sequencing; and
identifying mutations by clustering/aligning the sequences of the amplified products or fragments without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and wherein step (d) is immediately followed by step (e) such that no heteroduplex analysis is performed,(f) screening the identified mutations for a modified function of the target sequence;
(g) designing a primer directed to hybridize to the identified mutation;
(h) amplifying the pooled DNA of step (b) with the primer of step (g) and one of the primers of step (c), and;
(i) identifying the population member or members carrying the mutation.
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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
17 Claims
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1. A method for the detection of a mutation in a target sequence in a member of a mutagenized population, comprising the steps of:
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(a) isolating genomic DNA of each member of the mutagenized population to provide DNA samples of each population member; (b) pooling the DNA obtained in step (a); (c) amplifying the target sequence with a pair of primers from the DNA pools to obtain amplification products; (d) pooling the amplification products of step (c) to create a library of amplification products; (e) determining the nucleotide sequence of the amplification products or fragments of step (d) using high throughput sequencing; and
identifying mutations by clustering/aligning the sequences of the amplified products or fragments without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and wherein step (d) is immediately followed by step (e) such that no heteroduplex analysis is performed,(f) screening the identified mutations for a modified function of the target sequence; (g) designing a primer directed to hybridize to the identified mutation; (h) amplifying the pooled DNA of step (b) with the primer of step (g) and one of the primers of step (c), and; (i) identifying the population member or members carrying the mutation. - View Dependent Claims (3, 4, 5, 6, 8, 9, 13, 14, 15)
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2. A method for the detection of a mutation in a target sequence in a member of a mutagenized population, comprising the steps of:
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(a) isolating genomic DNA of each member of the mutagenized population to provide for DNA samples of each population member; (b) pooling the DNA obtained in step (a); (c) amplifying part of the target sequence with a pair of primers from the DNA pools to obtain amplification products; (d) pooling the amplification products of step (c) to create a library of amplification products; (e) determining the nucleotide sequence of the amplification products of step (d) using high throughput sequencing; and
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 wherein step (d) is immediately followed by step (e) such that no heteroduplex analysis is performed and(f) identifying members having the mutation. - View Dependent Claims (10, 11, 12, 16, 17)
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7. The method according to claim wherein sequencing is performed on a solid support such as a bead.
Specification