Brassica genomic assays
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.
1. A method of detecting and quantifying the amount of Brassica A genomic DNA in a sample, the method comprising:
(a) amplifying a genomic DNA fragment of the Brassica A genome with two or more oligonucleotide primer sequences selected from the group consisting of SEQ ID NOS: 21-24, 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, and 28, and wherein SEQ ID NOS: 25, 26, 27, and 28 of the amplified fragment identifies the presence of Brassica A genome in the sample; and
(b) detecting the amplified DNA fragment and quantifying the Brassica A genome from the amplified DNA fragment of the Brassica A genome.
2. 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.
3. The method of claim 1 , wherein the genomic DNA comprises a FatA gene.
4. 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.
5. A method of determining the relative amount of a Brassica transgenic event in a sample, the method comprising:
(a) performing a Brassica A genome specific polymerase chain reaction with two or more oligonucleotide primer sequences selected from the group consisting of SEQ ID NOS: 21-24, wherein the reaction includes 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, and 28;
(b) determining the total amount of Brassica A genomic DNA in the sample;
(c) performing an event specific assay for the transgenic event to determine the amount of the transgenic event in the sample; and
(d) comparing the amount of the transgenic event DNA to the total amount of Brassica A genomic DNA in the sample.
6. The method of claim 5 , wherein the genomic DNA comprises a FatA gene.
7. The method of claim 5 , wherein the determination comprises performing a quantitative polymerase chain reaction.
8. A method of determining an 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 two or more primers selected from the group consisting of SEQ ID NOS: 21-24, wherein the primers bind 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 and wherein the genomic region comprising one of SEQ ID NOS: 25-28 is amplified;
(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.
9. A method of establishing purity of a Brassica seed lot, the method comprising performing a polymerase chain reaction wherein the polymerase chain reaction comprises two or more oligonucleotide primers or probes capable of discriminating the Brassica A genome from the Brassica B and C genomes, wherein the oligonucleotide primers and/or probes is selected from the group comprising SEQ ID NOS: 21-24 and 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, and 28, and thereby quantifying Brassica A genome DNA in said Brassica seed lot.
10. The method of claim 9 , wherein the oligonucleotide primers and/or probes comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21, 22, 23, and 24.
11. A method of quantifying the amount of a transgenic element in a Brassica sample, the method comprising:
(a) performing a polymerase chain reaction wherein the polymerase chain reaction comprises two or more oligonucleotide primers or probes selected from the croup consisting of SEQ ID NOS: 21-24 that are capable of discriminating the Brassica A genome from the Brassica B and C genomes, the oligonucleotide primers or probes bind to a target region of the Brassica A genome comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 29, 30, and 31, and wherein the target Brassica A genome comprising one of SEQ ID NOS: 25-28 is amplified and thereby quantifying Brassica A genome DNA in the sample;
(b) performing a second polymerase chain reaction to detect the transgenic element present in the sample; and
(c) determining the relative amount of the transgenic element present in the Brassica sample by comparing amount of the transgenic element present in the sample to the amount of Brassica A genomic DNA in the sample.
12. A method of determining seed purity of a seed sample suspected of containing the Brassica A genome, the method comprising:
(a) specifically amplifying a genomic DNA of the Brassica A genome with two or more oligonucleotide primers selected from the group consisting of SEQ ID NOS: 21-24, wherein the amplified DNA comprises at least one of the nucleotide sequences of a genomic region selected from the group consisting of SEQ ID NOS: 25, 26, 27, and 28; and
(b) determining the purity of the seed sample based on the presence or absence of the Brassica A genome.
13. The method of claim 12 , wherein the seed sample contains at least one of broccoli, brussel sprouts, mustard seeds, cauliflower, collards, cabbage, kale, kohlrabi, leaf mustard, or rutabaga.
14. 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, and 28, optionally under high stringency conditions, and;
(b) detecting the presence and/or quantity of the Brassica A genome.
15. The method of claim 14 , 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.
16. The method of claim 14 , wherein the probe comprises the nucleotide sequences of SEQ ID NOS: 21, 22, 23, or 24.