Methods to determine zygosity in a bulked sample

A kind of sample, a specific technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, recombinant DNA technology, etc., can solve the problems of insensitivity and strength

Inactive Publication Date: 2013-11-20
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is insensitive and robust for detecting any hemizygous, null or any other unintentional contamination in completed seed batches

Method used

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  • Methods to determine zygosity in a bulked sample
  • Methods to determine zygosity in a bulked sample
  • Methods to determine zygosity in a bulked sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Plant material:

[0036] Parental Line Screening: To determine whether the border sequences at the transgene insertion site are highly conserved and whether event-specific primers can be used across various genetic backgrounds, a total of 92 diverse inbred lines representing different heterotic groups were screened ) and location (such as North America, South America, Europe), Stiff stalk, non-Stiff stalk, public and proprietary sources (Table 1).

[0037] Table 1: A batch of material used to screen border sequences for transgene insertion sites.

[0038]

[0039]

[0040]

[0041]

[0042] Bulk Seed Screening : To demonstrate the application of DNA-based tests to whole seed samples and to determine the sensitivity of the detection, known pure homozygous DAS-59122, heterozygous DAS-59122, and null (conventional) seeds were used Create the seed set mentioned below. Count these into 6 different 50mL Falcone tubes:

[0043] 1.100 homozygous DA...

Embodiment 2

[0049] Example 2: Seed Grinding and DNA Extraction

[0050] The seeds were finely ground and genomic DNA was isolated using the Qiagen DNEasy kit (Valencia, CA). Five separate genomic DNA extractions were done from each seed batch. Purified genomic DNA was quantified using the QuantIt Picogreen DNA kit and diluted to normalized concentrations.

Embodiment 3

[0051] Example 3: TaqMan-based zygosity assay:

[0052] Conjugation assays were performed using different sets of reagents consisting of different primer and probe sequences. Methods and reagents were designed specifically for the DAS-59122 event. A schematic diagram of the zygosity assay design is provided in Figure 1 . In addition to the two probes, the method utilizes a gene-specific primer, a wild-type primer, and a gene-specific / wild-type (common) primer. The probes consisted of wild-type-specific and transgene-specific probes. The first method incorporates all primers and probes within the same reaction ("single reaction method"). To improve the sensitivity of zygosity detection, an additional method was also tested in which two separate independent reactions were performed ( image 3 ) ("Multiple Reaction Method"). A set of wells contains a wild-type specific primer, a common primer, and a wild-type specific probe. Another set of wells contains transgene-specific ...

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Abstract

Methods of determining the presence or absence of an inserted nucleotide sequence at a particular insertion site in a nucleic acid include: isolating a nucleic acid from the bulked tissue sample; contacting the nucleic acid with a forward primer able to bind to the nucleic acid upstream of the insertion site, a first reverse primer specific for the inserted nucleotide sequence, and a second reverse primer able to bind to the nucleic acid downstream of the insertion site. The primers may be used to reproduce nucleic acids between the primers. The reproduced nucleic acids may be analyzed to determine if an inserted nucleotide sequence is present or absent in the sample.

Description

[0001] priority claim [0002] This application claims the benefit of the filing date of US Provisional Patent Application Serial No. 61 / 428,142, filed on December 29, 2010, for "Methods to Determine Zygosity in a Bulked Sample." Background of the Invention [0003] Completion of quality control testing for any contamination in the finished line is very important for successful hybrid seed production and for maintaining and building good business relationships with customers. Contamination during seed augmentation of completed lines can come from pollination of unintentional transgenic or non-transgenic plants grown near the production site or during seed processing, and more importantly, contamination due to pollen leakage from sterile plants. To ensure that the complete line is completely homozygous and does not have any hemizygous, empty or unintentional transgenic lines, the tester-row method is currently followed. The tester method uses ELISA technology to assess zygosit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6827C12Q1/6895C12Q1/6858C12Q1/686
Inventor C-S.钱那巴萨瓦拉迪亚
Owner DOW AGROSCIENCES LLC
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