Method to screen for a mutant within a population of organisms by applying a pooling and splitting approach
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A technology of organisms and species, applied in the direction of mutant preparation, microbial measurement/inspection, microbial library, etc., can solve unrealistic problems
Active Publication Date: 2019-03-15
CARLSBERG BREWERIES AS
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[0021] Finding predetermined and complex mutations using non-GM methods has so far been considered unrealistic
Also along this line, there is no hint as to what sample size would reliably identify a specific nucleotide substitution of interest
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Embodiment 1
[0484] - Example 1: Growing barley plants in "fields";
Embodiment 2
[0485] - Example 2: Harvesting of grain in a single "plot".
[0486] WS3: Determine if a library sample contains mutant grain
[0487] Step 3.1: Divide a single "Total Grain" grain pool into two parts
[0488] The threshed grain in a single "Total Grain" bag was divided into two parts, where the "Total Sub-Grain" sample consisted of ~1500 kernels (labeled SGT#01, SGT#02, etc.; see Figure 2C ; Action 1), its processing details refer to the following steps 3.2 to 3.4. Broadly speaking, grain processing in the "Total Grain Grain" section seeks to determine which fraction of the grain contains the mutation of interest. For reasons of clarity, but irrelevant to the associated actions of step 3, the remainder of the "total amount of grains" consists of ~4500 kernels, which are processed as detailed in the description relating to step 4.
[0489] For purposes of clarity of description, and not by way of limitation, the following Step 3.1 related subject matter is detailed in the ...
Embodiment 3
[0490] - Example 3: Description of the grains in "Total amount of sub-grains".
[0491] Step 3.2: Preparation of flour samples from the grains of the "Total Grain Grains"
[0492] Total samples of each sub-grain containing 1500 grains, ie samples designated as SGT#01, SGT#02, etc., were ground in a standard laboratory grinder (see Figure 2B , action 2), with thorough cleaning between applications, resulting in the corresponding flour samples - denoted as SFT#01, SFT#02, etc. (see Figure 2C ).
[0493] For purposes of clarity of description, and not by way of limitation, Step 3.2-related subject matter below is detailed in the following examples:
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Abstract
In traditional plant breeding approaches, chemical mutagenesis may be utilized to introduce nucleotide substitutions at random in the genome of a plant, i.e. without possibilities to control the sitesof nucleotide changes. Because of genome complexities, the statistical probability is extremely little when it comes to finding a predetermined nucleotide substitution. The present invention, however, demonstrates how a novel, alternative use of digital polymerase chain reaction (dPCR), preferably droplet dPCR (ddPCR), is developed to exploit finding of specific nucleotide substitutions in mutated genes. The entire platform comprises a screening method with a library of mutagenized organisms, digital PCR -based systems and a set-up to propagate and analyze identified, mutated organisms.
Description
field of invention [0001] The present invention provides highly accelerated methods and processes that can be scaled up in practice and handle the production, selection and / or propagation of organisms at one or more nucleotide (NOI) sites of interest have specific, predetermined mutations. For example, the methods of the invention can be used to increase the speed and ability to produce plants having specific, predetermined mutations in one or more NOIs. Background technique [0002] Genetic methods for generating genetically modified (GM) organisms (GMOs) are widely available. However, the application of GMOs is generally less desirable for many purposes, especially in the food and beverage industry. Therefore, there remains a long-felt need to provide improved and more precise methods for traditional crop breeding, including cereals such as barley, to simply obtain better tailored raw materials for developing and manufacturing new products. Similar disadvantages exist i...
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Application Information
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