Patent ID: 8614073
Filing Date: 2013-12-24
Classification: C12Q,G16B

Abstract:
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.