# US 2015/0147757 A1 · Brassica genomic assays

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- Number: US 2015/0147757 A1
- Document ID: US20150147757A1
- Status: Published application
- Kind: A1
- Application number: 14/402,485
- Filed: 2013-05-23
- Published: 2015-05-28
- Earliest claimed: 2012-05-23
- Assignees: E. I. Du Pont de Nemours and Company
- Applicants: E I Du Pont De Nemours and Company
- Inventors: Matthew Curtis Harmon; Nancy L. Henderson; Cathy Xiaoyan Zhong
- Web page: https://patentgrep.com/patent/US20150147757A1

## CPC

- C12Q 1/6895: for plants, fungi or algae
- C07K 14/415: from plants
- C12Q 2600/13: Plant traits
- C12Q 2600/158: Expression markers
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Methods and compositions for detecting, identifying, and quantifying Brassica A genomic DNA are described. The methods are specific to the Brassica A genome and do not cross-react with other Brassica species, crops or weedy relatives that could contribute to contamination of a canola field.

## Claims

### Claim 1

A method of detecting and quantifying the amount of Brassica A genomic DNA in a sample, the method comprising:
a) specifically amplifying a genomic DNA fragment of the Brassica A genome, wherein the amplified DNA fragment comprises at least one of the nucleotide sequences of a genomic region selected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and 35; and
b) detecting and quantifying the Brassica A genome from the amplified fragment of the Brassica A genome.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amplified fragment of Brassica A genome comprises (i) the nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or 31; or (ii) a nucleic acid fragment of at least one of SEQ ID NOS: 29, 30, or 31, wherein the nucleic acid fragment is selected from the group consisting of nucleotide position from about 50 to about nucleotide position 400, 50 to about nucleotide position 100, 50 to about nucleotide position 350, 400 to about nucleotide position 350, 400 to about nucleotide position 200, and 400 to about nucleotide position 100 of SEQ ID NOS: 29, 30, or 31.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amplification is performed with a primer pair comprising nucleotide sequences selected from the group consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24.

### Claim 4 (depends on 1)

The method of claim 1, wherein the genomic DNA comprises a FatA gene.

### Claim 5

(canceled)

### Claim 6

(canceled)

### Claim 7 (depends on 1)

The method of claim 1 wherein the amplified fragment of Brassica A genome comprises the nucleotide sequence of at least one of SEQ ID NOS. 29, 30, or 31 from nucleotide position about 50 to about 400.

### Claim 8

(canceled)

### Claim 9

(canceled)

### Claim 10

(canceled)

### Claim 11

A method of determining adventitious presence of a Brassica transgenic event in a sample, the method comprising:
a) obtaining a sample suspected of containing a Brassica transgenic event;
b) performing a Brassica A genome specific polymerase chain reaction, with a primer, wherein the primer binds to a genomic region of the Brassica A genome, the genomic region selected from the nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or 31 from nucleotide position about 50 to about 400;
c) determining the total amount of Brassica A genomic DNA in the sample;
d) performing an event specific quantitative assay for the transgenic event to determine the amount of the transgenic event DNA in the sample; and
e) comparing the amount of the transgenic event to the total amount of Brassica in the sample.

### Claim 12 (depends on 11)

The method of claim 11 wherein the primer is selected from the group consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24.

### Claim 13

An amplicon comprising at least one of the nucleotide sequences of a genomic region selected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and 35 wherein the amplicon is not larger than 500 base pairs.

### Claim 14

(canceled)

### Claim 15

A detection kit comprising oligonucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24 wherein the oligo is less than about 50 nucleotides and one or more reaction components to perform a quantitative reaction.

### Claim 16 (depends on 1)

A method of determining trait purity of a Brassica trait, the method comprising:
a) obtaining a sample of a Brassica trait; and
b) performing the Brassica A genome specific assay of claim 1.

### Claim 17 (depends on 16)

The method of claim 16, wherein the Brassica trait is selected from the group consisting of RT73, RT200, MON88302, DP-073496, HCN92, T45 (HCN28), 23-18-17, 23-198, OXY-235, MS1, MS3, MS6, MS8, RF1, RF2, RF3, and Topas 19/2.

### Claim 18 (depends on 16)

The method of claim 16, wherein the determination comprises performing a quantitative polymerase chain reaction.

### Claim 19

A method of establishing purity of a Brassica seed lot, the method comprising performing a polymerase chain reaction wherein the oligonucleotide primers or probes are capable of discriminating the Brassica A genome from the Brassica B and C genomes, wherein the oligonucleotide primers and/or probes bind to a target region of the Brassica A genome, the target region comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and 35.

### Claim 20 (depends on 19)

The method of claim 19, wherein the oligonucleotide primers and/or probes comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 19, 20, 21, 22, 23, and 24.

### Claim 21

(canceled)

### Claim 22

(canceled)

### Claim 23 (depends on 19)

The method of claim 19, wherein the seed lot contains seeds selected from the group consisting of broccoli, brussel sprouts, mustard seeds, cauliflower, collards, cabbage, kale, kohlrabi, leaf mustard, or rutabaga.

### Claim 24

A method of determining the presence and/or quantity of the Brassica A genome, the method comprising:
a) specifically hybridizing DNA of the Brassica A genome with a probe, wherein the probe selectively binds to at least one of the nucleotide sequences of a genomic region selected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and 35, optionally under high stringency conditions, and;
b) detecting the presence and/or quantity of the Brassica A genome.

### Claim 25 (depends on 24)

The method of claim 24, wherein the probe binds the genomic region of the Brassica A genome selected from the nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or 31 from nucleotide position about 50 to about 400.

### Claim 26 (depends on 24)

The method of claim 24, wherein the probe comprises the nucleotide sequences of SEQ ID NOS: 19, 20, 21, 22, 23, or 24.

### Claim 27

(canceled)