Detection of AAD-1 event DAS-40278-9

A technology of AAD-1 and events, applied to the determination/testing of microorganisms, enzymes, biochemical equipment and methods, etc., which can solve the problems of not showing the use of reference genes, etc.

Active Publication Date: 2012-07-11
CORTEVA AGRISCIENCE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of a reference gene is not shown

Method used

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  • Detection of AAD-1 event DAS-40278-9
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  • Detection of AAD-1 event DAS-40278-9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Event-specific Taqman assays

[0098]An event-specific Taqman assay was developed to detect the presence of maize event DAS-40278-9 and determine the zygosity status of plants in breeding populations. To develop event-specific assays, specific Taqman primers and probes were designed based on the DNA sequence located at the junction of the 5' insertion plant. For specific detection of DAS-40278-9, a 73-bp DNA fragment spanning the 5' integration junction was amplified using two specific primers. Amplification of this PCR product was measured by a target-specific MGB probe synthesized by Applied Biosystems containing a FAM reporter at its 5' end. The specificity of this Taqman assay for AAD-1 maize event DAS-40278-9 was tested against 16 different AAD-1 maize events and double-stranded forms of non-transgenic maize cultivars with a maize-specific endogenous reference gene, invertase .

Embodiment 11

[0099] Example 1.1: gDNA isolation

[0100] gDNA samples from 16 different AAD-1 maize events and non-transgenic maize varieties were tested in this study. Two methods, Qiagen kit or CTAB, were used to extract gDNA. For the extraction of gDNA samples with the Qiagen preparation kit, eight fresh maize leaves were used for gDNA extraction according to the protocol of the modified Qiagen DNeasy 96 Plant Kit. For gDNA samples extracted using the CTAB procedure, approximately 0.3 g of lyophilized leaf tissue was used following the protocol from Permingeat et al., 1998. gDNA was quantified using the Pico Green method according to the vendor's instructions (Molecular Probes, Eugene, OR). The gDNA samples were diluted with DNase-free water to obtain a concentration of 10 ng / μL for the purpose of this study.

Embodiment 12

[0101] Example 1.2: Taqman Assay and Results

[0102] Specific Taqman primers and probes were designed for the DAS-40278-9 event-specific Taqman assay. These reagents can be used to detect AAD-1 corn event DAS-40278-9 under the conditions listed in the table below. Table 1 lists the primer and probe sequences developed specifically for the detection of event DAS-40278-9.

[0103] Table 1: PCR Primers and Probes

[0104]

[0105]

[0106] Multiplex PCR conditions for amplification are as follows: 1X PCR buffer, .5-2.5mM MgCl 2 , .2mM dNTP, 0.2μM Primer Corn-278-F, 0.2μM Primer Corn-278-R, 0.2μM Primer_IV-F, 0.2μM Primer_IV-R, 0.08μM Probe_Corn-278-Probe, 0.08uM Probe_IV-Probe, 40U / mL HotStart Taq, 0.6 to 2.4ug / mL DNA, total reaction 25μl. Various concentrations of MgCl were tested 2 and DNA. The mixture was amplified using the following conditions: i) 95°C for 15 minutes, ii) 95°C for 20 seconds, iii) 60°C for 60 seconds, iv) steps ii-iii repeated for 50 cycles, ...

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Abstract

This invention relates in part to detecting herbicide tolerant plants - more specifically, an aad-1 transformation event in corn plants. The subject invention also provides assays for detecting the presence of the subject event in a sample (of corn grain, for example). Kits and conditions useful in conducting the assays are also provided. The subject invention also relates in part to plant breeding using the subject methods. In some embodiments, said event / polynucleotide sequence can be "stacked" with other traits. More specifically, the invention relates in part to an endpoint TaqMan PCR assay for AAD-1 corn event 40278-9. Some embodiments are directed to assays that are capable of high throughput zygosity analysis. The subject invention further relates, in part, to the use of a preferred reference gene for use in determining zygosity.

Description

Background of the invention [0001] The aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1) protein. This trait confers herbicide resistance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly known as "fop" herbicides such as diclofop and quizalofop). Tolerance to pesticides and can be used as selectable markers during plant transformation and in breeding nurseries. The aad-1 gene itself was first disclosed in WO 2005 / 107437 (see also US 2009-0093366) as being involved in herbicide tolerance in plants. [0002] Various methods of event detection are known. However, they all have problems. One method is the Pyrosequencing technique as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, oligonucleotides are designed that overlap adjacent genomic DNA and insert DNA junctions. The oligonucleotides were hybridized to single-stranded PCR products from the region of interest (one pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12N9/0069C12N15/8274C12N9/0071
Inventor Y.C.崔T.W.格林S.诺瓦克N.周
Owner CORTEVA AGRISCIENCE LLC
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