ISOLATION OF MEGABASE-SIZED DNA FROM PLANT AND ANIMAL TISSUES
Disclosed herein are methods for isolation of long DNA molecules, for example megabase-sized genomic DNA molecules, from a biological sample, for example plant and animal tissues.
1 . A method for isolating DNA from a biological sample, the method comprising:
(a) homogenizing the biological sample to generate a homogenate;
(b) contacting the homogenate with a DNA and/or protein precipitating agent;
(c) embedding the homogenate in a porous matrix; and
(d) recovering DNA from the homogenate.
2 . The method of claim 1 , wherein cellular integrity, nuclear integrity, or both in the biological sample is at least partially maintained during step (a).
3 . The method of claim 1 , wherein the DNA and/or protein precipitating agent comprises ethyl alcohol, methyl alcohol, isopropyl alcohol, acetone, or any combination thereof.
4 . The method of claim 1 , wherein embedding the homogenate in the porous matrix comprises dispersing the homogenate throughout the matrix.
5 . The method of claim 1 , further comprising contacting the homogenate, following step (b), with a collagenase, an elastase, a lipase, an amylase, a hyaluronidase, an RNase, a fibornectinase, a lamininase, a protease, or any combination thereof.
6 . The method of claim 1 , wherein the biological sample comprises a plant tissue and wherein homogenizing the biological sample comprises treating the biological sample with a mechanical means, an enzymatic means, or a combination thereof.
7 . The method of claim 1 , further comprising contacting the homogenate before, after, or during step (b) with a crosslinking agent.
8 . The method of claim 6 , further comprising contacting the homogenate before, after, or during step (b) with a crosslinking agent.
9 . The method of claim 1 , further comprising separating one or more discrete DNA-containing entities from tissue fragments, intact cells, and cell remnants before step (c) and after step (b).
10 . The method of claim 1 , wherein the biological sample comprises a plant tissue, an animal tissue, or both.
11 . The method of claim 1 , wherein at least 30% of the DNA recovered from the homogenate is more than 20 kilobases.
12 . The method of claim 1 , wherein recovering DNA from the homogenate comprises treating the porous matrix embedded with the homogenate with an agent to remove non-DNA components.
13 . The method of claim 1 , wherein recovering DNA from the homogenate comprises contacting the porous matrix embedded with the homogenate with an elastase, a collagenase, hyaluronidase, an RNase, a fibornectinase, a lamininases, a lipase, a carbohydratase, a pectinase, a pectolyase, an amylase, an RNase, a hyaluronidases, or any combination thereof.
14 . A method for isolating DNA from a biological sample, the method comprising:
(a) contacting the biological sample with a DNA and/or protein precipitating agent;
(b) homogenizing the biological sample after step (a) to generate a homogenate;
(c) embedding the homogenate in a porous matrix; and
(d) recovering DNA from the homogenate.
15 . The method of claim 14 , wherein the DNA and/or protein precipitating agent is ethyl alcohol, methyl alcohol, isopropyl alcohol, or acetone.
16 . The method of claim 14 , further comprising contacting the homogenate, following step (b), with a collagenase, an elastase, a lipase, an amylase, a hyaluronidase, an RNase, a fibornectinase, a lamininase, a protease, or a combination thereof.
17 . The method of claim 14 , further comprising contacting the homogenate before, after, or during step (a) with a crosslinking agent.
18 . The method of claim 14 , further comprising contacting the homogenate after, or during step (b) with a DNA and/or protein precipitating agent.
19 . The method of claim 14 , wherein the biological sample comprises a plant tissue, an animal tissue, or both.
20 . The method of claim 14 , wherein at least 30% of the DNA recovered from the homogenate is more than 20 kilobases.