DNA extraction from seeds using osmoticum

Patent metadata

Field	Value
Publication number	US-9719893-B2
Application number	US-201214359238-A
Country	US
Kind code	B2
Filing date	Nov 28, 2012
Priority date	Nov 28, 2011
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

The invention relates to methods of extracting DNA from seeds, said method comprising pretreating said seeds by soaking the seeds in a pretreatment solution comprising an alkali in a concentration sufficient to soften said seed; crushing said seeds; extracting said DNA from said crushed seeds. Methods also relate to the use of pretreatment solutions which further comprise an osmoticum. A method of fragmenting plant material such as seed, a method of recovering extraction medium from seed fragmentation and a process of extracting a seed component from crushed seed material are also described.

First claim

The invention claimed is:

1. A method for extracting and isolating DNA from corn seeds, said method comprising:
a. pretreating said seeds by soaking the seeds in a pretreatment solution comprising:
i. an alkali in a concentration sufficient to soften said seed and
ii. an osmoticum at a concentration in the range of about 1 M to about 5 M, wherein said osmoticum is an aqueous solution of a solute selected from the group consisting of sodium chloride, potassium chloride, a polyethylene glycol (PEG), mannitol, a sugar alcohol, and sucrose;

b. crushing said seeds;
c. contacting a DNA extraction buffer with said crushed seeds, whereby said contacting extracts DNA from said crushed seed; and
d. the DNA solution is isolated and the DNA separated therefrom.

2. The method of claim 1 wherein said alkali is a solution comprising sodium hydroxide or potassium hydroxide.

3. The method of claim 2 wherein said alkali is present at a concentration of about 0.1 M to about 1 M.

4. The method of claim 1 wherein said soaking is continued for a period of time sufficient to soften said seed.

5. The method of claim 1 wherein said soaking is performed at a temperature between about 22° C. and about 65° C.

6. The method of claim 1 wherein said crushing is performed by a method comprising placing said seeds in a confined space and shaking them with size-reducing elements.

7. The method of claim 6 wherein said size-reducing elements are objects have sufficient hardness to break up the seeds.

8. A method for extracting and isolating DNA from corn seeds, said method comprising:
a. pretreating said seeds by soaking the seeds in a pretreatment solution comprising:
i. an alkali in a concentration sufficient to soften said seed and optionally
ii. an osmoticum at a concentration in the range of about 1 M to about 5 M, wherein said osmoticum is an aqueous solution of a solute selected from the group consisting of sodium chloride, potassium chloride, a polyethylene glycol (PEG), mannitol, a sugar alcohol, and sucrose;

b. crushing said seeds, wherein said crushing is performed by a method comprising placing said seeds in a confined space and shaking them with size-reducing elements;
c. contacting said crushed seeds with a DNA extraction buffer; and
d. the DNA solution is isolated and the DNA separated therefrom.

Assignees

• Syngenta Participations Ag

Inventors

• Happappel Ulrich

Classifications

• G01N2001/2866

  Grinding or homogeneising · CPC title

• B02C9/00

  Other milling methods or mills specially adapted for grain · CPC title

• G01N1/286 Primary

  involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F) · CPC title

• B02C1/14

  Stamping mills · CPC title

• B02C23/18

  Adding fluid, other than for crushing or disintegrating by fluid energy ({for tumbling mills B02C17/186;} feeding devices B02C23/02) · CPC title