HIGH THROUGHPUT SCREENING OF MUTAGENIZED POPULATIONS

• US 20090170713A1
• Filed: 09/21/2006
• Published: 07/02/2009
• Est. Priority Date: 09/29/2005
• Status: Abandoned Application

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: van Tunen, Adrianus Johannes, van Eijk, Michael Josephus Theresia
• Application Number:
  US12/088,794
• Publication Number:
  US 20090170713A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

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.

Claims

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 (optionally labeled) primers from the DNA pools;

(d) pooling the amplification products of step (c) to create a library of amplification products;

(e) optionally, fragmenting the amplification products in the library;

(f) determining the nucleotide sequence of the amplified products or fragments using high throughput sequencing;

(g) identifying mutations by clustering/aligning the sequences of the fragments;

(h) screening the identified mutations for a modified function of the target sequence;

(i) designing a primer directed to hybridize to the identified mutation;

(j) amplifying the library of step (d) with the primer of step (i) and one of the primers of step (c);

(k) identify the population member(s) carrying the mutation;

(l) optionally, confirming the mutation by amplifying the target sequence from the member(s) of step (k) using the primers of step (c) and determining the sequence of the amplified product.

3. The method according to claim 1, wherein the mutagenized population is obtained by treating the genome with one or more selected from the group consisting of mutation-inducing chemicals, ionizing radiation, targeted nucleotide exchange or region targeted muatgenesis.

4. The method according to claim 1, wherein the population is not mutagenized but comprises sub-populations that contain naturally occurring mutations.

5. The method according to claim 1, wherein the pooling is a 3D-pooling strategy.

6. The method according to claim 1, wherein the high throughput sequencing is performed by synthesis, preferably pyrosequencing.

7. The method according to claim 1, wherein sequencing is performed on a solid support such as a bead.

8. The method according to claim 1, wherein the sequencing is based on High Throughput Sequencing, preferably Sequencing-by-Synthesis.

9. The method according to claim 1, wherein the sequencing is performed by synthesis, preferably pyrosequencing.

10. The method according to claim 6, wherein sequencing comprises the steps of:

(i) ligating sequencing adaptors to the fragments;

(ii) annealing said adaptor-ligated fragments to beads, each bead annealing with a single fragment;

(iii) emulsifying the beads in water-in-oil microreactors such that each microreactor contains a single bead;

(iv) performing emulsion PCR to amplify adaptor-ligated fragments on the surface of the beads

(v) selecting/enriching beads to which are attached said amplified adaptor-ligated fragments

(vi) placing the beads in wells such that each well comprises a single bead; and

(vii) generating a pyrophosphate signal.

11. The method according to claim 1, wherein the primers in steps (c) and/or step (i) contain nucleotides with improved binding affinity.

2. 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 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 tagged (optionally labeled) primers from the DNA pools, wherein at least one of the primers preferably comprises a gene-specific section, a tag and a sequence primer binding site;

(d) pooling the amplification products of step (c) to create a library of amplification products;

(e) determining the nucleotide sequence of the amplification products using high throughput sequencing;

(f) identifying mutations by clustering/aligning the sequences of the fragments;

(g) identifying the member(s) having the mutation using the tags;

(h) optionally, confirming the mutation by amplifying the target sequence from the member(s) of step (g) using the primers of step (c) and determining the sequence of the amplified product.

12. The method according to claim 2, wherein the part of the target sequence that is amplified in step (c) is from about 80 to about 400 bp.

13. The method according to claim 12, wherein the part of the target sequence that is amplified in step (c) is from about 90 to about 300 bp.

14. The method according to claim 12, wherein the part of the target sequence that is amplified in step (c) is from 100 to about 200 bp.

15. Kit comprising one or more (labeled) primers for a particular gene or trait.

16. Kit according to claim 15, further comprising mutation- or allele-specific primers.

17. Kit according to claim 15 further comprising beads and/or sequencing primers.