Endpoint TAQMAN methods for determining zygosity of corn comprising TC1507 events
A TC1507, event technology, applied in the ment field, can solve problems such as not using reference genes
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Embodiment 1
[0087] Example 1: Isolation and quantification of total genomic DNA and PCR primer amplification. Genomic DNA from Cry1F homozygous, hemizygous and wild-type samples was isolated from individual samples by punching 8 leaf discs per sample and grinding the discs to a fine powder using a Genogrinder 2000. use custom DNA was extracted using the gDNA Plant Kit (Invitrogen, Carlsbad, CA) or Qiagen DNeasy 96-well kit (Valencia, CA). Before PCR, use Quant-iT TM DNA samples were quantified using a quantification kit (Invitrogen, Carlsbad, CA) using the manufacturer's instructions.
[0088] Oligonucleotide primers and dual-labeled TaqMan probe with FAM and Black Hole Quencher 1 (BHQ1) were synthesized by MWG Biotech (High Point, NC) (Table 1b).
[0089] Table 1b: Sequences of primers and dual-labeled TaqMan probes
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[0092] Dual-labeled TaqMan probes with Cy5 and BHQ2 were synthesized by IDT (Integrated DNA Technologies, Coralville, IA). All primers were ...
Embodiment 2
[0109] Example 2: PCR efficiency test of maize endogenous genes. One aspect of developing an endpoint TaqMan zygosity assay is the selection of the most appropriate endogenous gene as a reference gene. We selected species-specific invertases with low copy numbers in the genome, ie a suitable reference gene. Four maize endogenous genes were initially investigated among thousands of possibilities, namely alcohol dehydrogenase 1 (adh1), hypermobility group a (hmga), invertase (ivr), and zein (zein) As a possible reference gene for maize Cry1F in event TC1507.
[0110] The process of selecting an appropriate reference gene involved first combining 30 ng of extracted Cry1F homozygous, hemizygous and wild-type maize genomic DNA controls (extracted according to the isolation protocol described in this example) to assess the PCR efficiency of all primers. PCRs for TC1507 and the 5 initially selected reference genes (ivr, ivr104, adh, hmg, zein) were set up according to Table 2c. T...
Embodiment 3
[0118] Example 3: Testing of an endpoint TaqMan assay for zygosity genotyping. Multiplexing of one reference gene (ivr, ivr104, hmg or zein) with TC1507 was tested using endpoint TaqMan PCR (Table 3) using a Cry1F single stack population with segregating TC1507 (Q:07K:PF04DS_ZYGO) . DNA was normalized to 10 ng / μl prior to endpoint TaqMand PCR. TaqMan PCR reactions were terminated at 28 cycles and then measured with a spectrofluorometer. Calculated above background (H 2 O) of FAM (TC1507) (as signal above background 1 (SOB1)), and the fluorescent signal of Cy5 (reference gene) 2 (SOB2) above background. Plot the SOB1 / SOB2 ratio as a scatter plot in Excel. In segregating populations, three clusters of data points should be obtained, allowing for visual determination of cut-off points. It was found that Ivr104 multiplexed with TC1507 only under the reaction conditions disclosed herein can produce unambiguous genotype calls. Other initially selected reference gene responses...
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